Non-viral compositions and methods for transfecting gut cells in vivo

ABSTRACT

The present invention provides chitosan-based nanoparticles that can protect nucleic acids and deliver the same into gut mucosal cells. Compositions and methods for the expression of therapeutic nucleic acids in cells of the gut mucosa are provided. Compositions and methods for delivering therapeutic proteins systemically from cells of the gut mucosa are also provided.

FIELD

The invention relates to chitosan-based nanoparticles and nucleic acidcarriers. Additionally the invention relates to methods of transfectinggut cells in vivo.

BACKGROUND

Chitosan is a non-toxic cationic copolymer of N-acetyl-D-glucosamine andD-glucosamine that possesses favorable mucosal adhesion properties andhas been widely used in controlled drug delivery. The mucoadhesivity ofchitosan is thought to prolong the residence time of an associated drugin the gastrointestinal tract, thereby increasing its bioavailability.(Kotze A F, Luessen H L, Thanou M, Verhoef J C, de Boer A G, Juninger HE, Lehr C M. Chitosan and chitosan derivatives as absorption enhancersfor peptide drugs across mucosal epithelia. In: Mathiowitz E, ChickeringD E, Lehr C M, eds. Bioadhesive Drug Delivery Systems. New York, N.Y.:Marcel Dekker; 1999.)

Several groups have explored the potential of chitosan as a DNA deliveryvehicle, and the properties of a number of chitosan/DNA complexes havebeen examined in an attempt to identify compositions well suited forgene transfection. The complexes have been found to vary in, among otherproperties, solubility, propensity for aggregation, complex stability,particle size, ability to release DNA, and transfection efficiency.Chitosans of large molecular weight are relatively insoluble atphysiological pH and are dissolved in acidic solution for use. Onceformed, complexes containing large molecular weight chitosan and DNA arerelatively stable, but poor transfection efficiencies have beenreported, possibly owing to poor uptake and release of DNA. Lowmolecular weight chitosan/DNA complexes are more soluble but less stablein solution. Chitosan polymers of low molecular weight reportedly formunstable complexes with DNA, as indicated by separation in an electricfield (agarose gel electrophoresis). Such complexes also show lowtransfection efficiencies and low levels of reporter gene expression invitro (e.g., Koping-Hoggard et al., Gene Ther., 8:1108-1121, 2001;MacLaughlin, et al., J Control Release. 1998 Dec. 4; 56(1-3):259-72;Sato et al., Biomaterials, 22:2075-2080, 2001; US 2005/0170355; US2005/0164964). Experiments have also shown that low molecular weightchitosan/DNA complexes tend to release DNA in response to challenge withsalt or serum, suggesting they are poorly suited to many in vivoapplications.

Studies of the effect of chitosan molecular weight on transfectionefficiency in vitro have been equivocal. Some studies have shown nosignificant dependence on molecular weight for chitosan polymers in thesize range of 20-200 kDa (Koping-Hoggard et al., supra; MacLaughlin etal, supra). However, others (Sato et al., supra) have reported thatchitosans of 15 kDa and 52 kDa show higher reporter gene expression invitro than chitosan polymers >100 kDa and chitosan polymers of 1.3 kDaare ineffective. Moreover, discordance between in vitro and in vivotransfection efficiencies has been frequently reported (e.g.,Koping-Hoggard et al., Gene Ther. 2004 October; 11(19):1441-52; US2005/0170355; US 2005/0164964).

A number of studies have examined the ability of large molecular weightchitosan/DNA complexes to transduce cells of the gastrointestinal tract.Chitosan/DNA complexes incorporating an endosomolytic peptide have beenadministered directly to the upper small intestine and colon have beenshown to produce reporter gene expression in epithelial cells, Peyer'spatches and mesenteric lymph nodes (MacLaughlin et al., supra).Additionally, oral administration of a chitosan complex with DNAencoding erythropoietin has been shown to produce a transient hematocritincrease (Chen et al., World J. Gastroenter., 10:112-116, 2004).Chitosan has also been used in a food allergy model to orally deliverDNA encoding a peanut allergen protein, Arah2, in an attempt to rendermice tolerant to the ingestion of peanut extract (Roy et al., Nat. Med.,5:387-391, 1999). In all, relatively low transfection efficiencies andlow levels of transgene expression have been reported, due in part topoor DNA uptake and release, but also due in part to rapid turnover ofcells in the gut. The gut epithelium is one of the most rapidly renewingtissues in the body, with epithelial cell turnover every 3-5 days.Importantly, transgene expression in mucosal cells of the gut isadditionally complicated by the fact that luminal mucosal cells areshort-lived, providing a brief period of time for expression of DNA onceit has entered the nucleus of a cell.

Other research groups have chemically modified chitosan in a variety ofways in efforts to develop DNA complexes with improved transfectionefficiencies and other desirable properties, such as the ability totransduce distal gut tissue. Kai et al. point out in their report thatchitosan/DNA compositions that have left the stomach and entered themore neutral environment of the duodenum lose positive charge with theattendant shift in pH, and consequently tend to release associated DNA.Kai et al. report that N-acetylation of lyophilized large molecularweight chitosan/DNA complexes stabilizes orally delivered complexes andincreases efficiency of distal gut transduction. Kai et al., Pharm. Res.21:838-843, 2004.

Given the lifespan of luminal gut mucosal cells, it is perhaps notsurprising that long-term expression of genes delivered to gut mucosa bychitosan has not been reported. Additionally, the ability of chitosanDNA complexes to transfect less prevalent cell types of the gut mucosa,such as endocrine cells, has not been examined in detail. The difficultyassociated with achieving long-term expression and transfection of gutendocrine cells may be appreciated by a consideration of gut structure.

The wall of the gastrointestinal canal is composed of four layers. Theinnermost layer of the canal is the mucosal layer, which is composed ofa lining epithelium that borders the lumen. The epithelium is the siteat which the body interacts with ingested materials. In areas of thegastrointestinal canal where absorption is effected, the epithelium is asingle cell in thickness. The epithelium rests on a basal lamina, whichin turn overlies the lamina propria. Beds of blood capillaries aredensely packed in the length of lamina propria underlying absorptiveregions of the canal, and it is into these vessels that the processedproducts of absorbed food matter pass.

The human small intestine consists of three portions: duodenum, jejunum,and ileum. The mucosa of the small intestine is extensively foldedgiving it a ruffled appearance as circular folds project into theintestinal lumen. Such folding fills a substantial area of theintestinal canal and increases the absorptive surface area of theepithelium by several fold. At the luminal surface, the folds of theintestinal mucosa present villi, which are evaginations of the mucosathat further increase absorptive area. Each villus, in turn, is coveredby an epithelium one cell thick. This epithelium is overwhelminglypopulated by absorptive enterocytes, which display thousands of shortmicrovilli on their apical (luminal) surface, increasing the absorptivearea many fold again. The outer surface of the microvilli, referred toas the glycocalyx, is filamentous and rich in carbohydrates. Thismembrane region is also rich in a wide variety of enzymes and transportsystems facilitating the breakdown and uptake of ingested material.

Absorptive enterocytes constitute greater than 90% of the epithelialcells of the villus, and an even greater proportion of the luminalsurface area. Scattered among these cells are the relatively smallnumber of enteroendocrine cells, which reportedly constituteapproximately 0.3% of the villus epithelium. In contrast to absorptiveenterocytes, which present a large and ultrastructurally complex apicalsurface, endocrine cells have broad basal surfaces juxtaposed tocapillaries of the lamina propria and narrow superiorly toward thelumen.

Even more elusive than the endocrine cells of the gut mucosa are the gutmucosal precursor cells. Inferior to the projecting villi, in theepithelium lining the depth of the crypts, lie precursor cells that giverise to the major cell types of the mucosa, including absorptiveenterocytes and gut endocrine cells. The villus epithelial layer forms acontinuous sheet of short-lived differentiated epithelial cells that isrenewed about every three days, and maintenance of the epitheliumrequires an enormous amount of cell division and differentiation.Precursor cells of the crypts generate progeny that migrate out of thecrypts toward villi and undergo differentiation.

Gut endocrine cells are generally characterized by their ability tosecrete a synthesized protein into the blood in response to a signal orstimuli (a “secretagogue”). Particular examples of endocrine cellsinclude K cells, L-cells, S-cells, G-cells, D-cells, I-cells, Mo-cells,Gr-cells.

K cells are located primarily in the stomach, duodenum, and jejunum.These endocrine cells secrete the hormone GIP, which normally functionsto potentiate insulin release after a meal.

Gut endocrine cells generally, and K cells in particular, are attractivecellular targets for the delivery of transgenes. These cells possess theability to process proforms of many proteins, and possess the cellularmachinery that provides for regulated secretion of protein into thesystemic circulation in response to cues. These properties have beenexploited previously (see Cheung et al., Science, 290:1959-1962, 2000;U.S. patent application Ser. No. 09/804,409; expressly incorporatedherein by reference). K cells engineered with a K-cell specificglucose-responsive insulin expression construct were observed to expressand secrete insulin in response to elevated blood glucose, and werecapable of restoring normal glucose tolerance in a mouse model ofdiabetes.

Despite a general interest in chitosan as an alternative to viral meansof nucleic acid delivery, low transfection efficiency, stability andsolubility problems, in vivo unpredictability, and an inability totransfect other than short-lived mucosal cells has largely preventedapplication of chitosan as a nucleic acid carrier in the harshenvironment of the gut.

SUMMARY OF INVENTION

In one aspect, the invention provides chitosan-based nanoparticlescomprising therapeutic nucleic acids that are capable of exerting atherapeutic effect when expressed in gut mucosal cells. Suchchitosan-based nanoparticles provide for novel non-viral methods oftreating or preventing a wide variety of conditions and disorders.

Disclosed herein are chitosan-based nanoparticles capable of producinglong-term expression of therapeutic nucleic acids in gut mucosa in vivo.Such nanoparticles are useful for the long-term production oftherapeutic RNAs or therapeutic proteins in gut mucosa.

Disclosed herein are chitosan-based nanoparticles capable of producingincreases in the systemic levels of therapeutic proteins encoded bytherapeutic nucleic acids. Such chitosan-based nanoparticles are capableof delivering physiologically relevant levels of therapeutic proteinsinto the blood circulatory system.

Disclosed herein are chitosan-based nanoparticles capable oftransfecting gut mucosal precursor cells in vivo. Such nanoparticles areuseful for the long-term production of therapeutic RNAs or therapeuticproteins in gut mucosa.

Disclosed herein are chitosan-based nanoparticles capable oftransfecting gut endocrine cell precursor cells in vivo. Suchnanoparticles provide for the production of therapeutic proteins in amucosal cell type that is capable of processing proproteins andsecreting therapeutic proteins in a regulated or constitutive mannerinto the systemic circulation.

In a number of preferred embodiments of the invention, chitosan-basednanoparticles comprise therapeutic nucleic acids encoding therapeuticproteins capable of exerting a therapeutic effect in non-gut tissue.Especially preferred are therapeutic proteins that have systemicactivity.

In a number of preferred embodiments of the invention, chitosan-basednanoparticles are engineered for the non-constitutive expression oftherapeutic nucleic acids in gut mucosal cells. Such nanoparticlesprovide for dynamic long-term expression of therapeutic nucleic acids.In a number of preferred embodiments, nanoparticles are engineered so asto provide for regulatable expression of therapeutic nucleic acids ingut mucosal cells.

In a number of preferred embodiments, nanoparticles are engineered so asto provide for the regulated secretion of therapeutic proteins from gutendocrine cells in response to a secretagogue.

In accordance with the objectives stated above, in one aspect, theinvention provides compositions comprising chitosan-based nanoparticles.

In one embodiment, the invention provides a composition comprising achitosan-based nanoparticle, which nanoparticle comprises (i) aplurality of chitosan polymers having an average molecular weightbetween 3 kDa and 250 kDa, and (ii) a therapeutic construct, wherein thetherapeutic construct comprises a therapeutic nucleic acid operablylinked to an expression control region that is functional in a gutmucosal cell, wherein the therapeutic nucleic acid is capable ofexerting a therapeutic effect when expressed in the gut mucosal cell,and wherein the chitosan-based nanoparticle is capable of effectingexpression of the therapeutic nucleic acid in gut mucosa for longer thanabout 4 days, more preferably for longer than about 5 days, morepreferably for longer than about 6 days, more preferably for longer thanabout 7 days, more preferably for longer than about 10 days, morepreferably for longer than about 2 weeks, more preferably for longerthan about 3 weeks, more preferably for longer than about 4 weeks, morepreferably for longer than about 6 weeks, more preferably for longerthan about 8 weeks, more preferably for longer than about 10 weeks, andmost preferably for longer than about 12 weeks.

In one embodiment, the invention provides a composition comprising achitosan-based nanoparticle, which nanoparticle is capable oftransfecting a gut mucosal precursor cell in vivo. Such a nanoparticlecomprises (i) a plurality of chitosan polymers having an averagemolecular weight between 3 kDa and 250 kDa, and (ii) a therapeuticconstruct, wherein the therapeutic construct comprises a therapeuticnucleic acid operably linked to an expression control region that isfunctional in a gut mucosal cell, wherein the therapeutic nucleic acidis capable of exerting a therapeutic effect when expressed in the gutmucosal cell. In a preferred embodiment, the nanoparticle is capable ofproducing expression of the therapeutic nucleic acid in gut mucosa forlonger than about 4 days, more preferably for longer than about 5 days,more preferably for longer than about 6 days, more preferably for longerthan about 7 days, more preferably for longer than about 10 days, morepreferably for longer than about 2 weeks, more preferably for longerthan about 3 weeks, more preferably for longer than about 4 weeks, morepreferably for longer than about 6 weeks, more preferably for longerthan about 8 weeks, more preferably for longer than about 10 weeks, andmost preferably for longer than about 12 weeks.

In one embodiment, the invention provides a composition comprising achitosan-based nanoparticle, which nanoparticle is capable of increasingthe systemic level of a therapeutic protein. Such a nanoparticlecomprises (i) a plurality of chitosan polymers having an averagemolecular weight between 3 kDa and 250 kDa, and (ii) a therapeuticconstruct, wherein the therapeutic construct comprises a therapeuticnucleic acid operably linked to an expression control region that isfunctional in a gut mucosal cell, wherein the therapeutic nucleic acidis capable of exerting a therapeutic effect when expressed in the gutmucosal cell, wherein the therapeutic nucleic acid encodes a therapeuticprotein that is delivered into the systemic circulation from the gutmucosa. In a preferred embodiment, the nanoparticle is capable ofproducing expression of the therapeutic nucleic acid in gut mucosa forlonger than about 4 days, more preferably for longer than about 5 days,more preferably for longer than about 6 days, more preferably for longerthan about 7 days, more preferably for longer than about 10 days, morepreferably for longer than about 2 weeks, more preferably for longerthan about 3 weeks, more preferably for longer than about 4 weeks, morepreferably for longer than about 6 weeks, more preferably for longerthan about 8 weeks, more preferably for longer than about 10 weeks, andmost preferably for longer than about 12 weeks.

In one embodiment, the nanoparticle consists essentially of (i) aplurality of chitosan polymers having an average molecular weightbetween 3 kDa and 250 kDa, and (ii) a therapeutic construct, wherein thetherapeutic construct comprises a therapeutic nucleic acid operablylinked to an expression control region that is functional in a gutmucosal cell, wherein the therapeutic nucleic acid is capable ofexerting a therapeutic effect when expressed in the gut mucosal cell.

In a preferred embodiment, the expression control region of achitosan-based nanoparticle of the invention has non-constitutiveactivity, and the nanoparticle is capable of producing long-term dynamicexpression of the therapeutic nucleic acid. In an especially preferredembodiment, the expression control region is regulatable, and thenanoparticle is capable of producing long-term regulatable expression ofthe therapeutic nucleic acid.

The chitosan polymers of chitosan-based nanoparticles preferably have anaverage molecular weight of less than about 250 kDa, more preferablyless than 230 kDa, more preferably less than 220 kDa.

In another preferred embodiment, the plurality of chitosan polymers ofthe nanoparticle have an average molecular weight from about 3 kDa toabout 210 kDa.

In another preferred embodiment, the plurality of chitosan polymers ofthe nanoparticle have an average molecular weight from about 10 kDa toabout 250 kDa.

In another preferred embodiment, the plurality of chitosan polymers ofthe nanoparticle have an average molecular weight from about 10 kDa toabout 210 kDa.

In another preferred embodiment, the plurality of chitosan polymers ofthe nanoparticle have an average molecular weight from about 3 kDa toabout 50 kDa.

In another preferred embodiment, the plurality of chitosan polymers ofthe nanoparticle have an average molecular weight from about 3 kDa toabout 6 kDa.

In another preferred embodiment, the plurality of chitosan polymers ofthe nanoparticle have an average molecular weight from about 200 kDa toabout 210 kDa.

In a preferred embodiment, the nanoparticle has an amine:phosphate (N:P)ratio from about 1:1 to about 100:1.

In another preferred embodiment, the nanoparticle has an N:P ratio fromabout 1:1 to about 6:1.

In another preferred embodiment, the nanoparticle has an N:P ratio fromabout 1:1 to about 4:1.

In another preferred embodiment, the nanoparticle has an N:P ratio ofabout 3:1.

In another preferred embodiment, the nanoparticle has an N:P ratio fromabout 10:1 to about 90:1.

In another preferred embodiment, the nanoparticle has an N:P ratio fromabout 10:1 to about 50:1.

In another preferred embodiment, the nanoparticle has an N:P ratio fromabout 10:1 to about 40:1.

In another preferred embodiment, the nanoparticle has an N:P ratio fromabout 10:1 to about 30:1.

In another preferred embodiment, the nanoparticle has an N:P ratio fromabout 60:1 to about 100:1.

In another preferred embodiment, the nanoparticle has an N:P ratio fromabout 70:1 to about 100:1.

In another preferred embodiment, the nanoparticle has an N:P ratio ofabout 20:1.

In another preferred embodiment, the nanoparticle has an N:P ratio ofabout 60:1.

In a preferred embodiment, the nanoparticle has a chitosan:nucleic acidw/w ratio from about 1:1 to about 50:1.

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio from about 1:1 to about 5:1.

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio from about 1:1 to about 3:1.

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio of about 2:1

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio from about 5:1 to about 45:1.

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio from about 5:1 to about 25:1.

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio from about 5:1 to about 20:1.

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio from about 5:1 to about 15:1.

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio from about 30:1 to about 50:1.

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio from about 35:1 to about 50:1.

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio of about 10:1

In another preferred embodiment, the nanoparticle has a chitosan:nucleicacid w/w ratio of about 30:1

In a preferred embodiment, the composition comprises nanoparticleshaving an average zeta potential between +5 mV and +50 mV at a pH of 5.

In another preferred preferred embodiment, the composition comprisesnanoparticles having an average zeta potential between +30 mV and +50 mVat a pH of 5.

In another preferred preferred embodiment, the composition comprisesnanoparticles having an average zeta potential between +30 mV and +40 mVat a pH of 5.

In another preferred embodiment, the composition comprises nanoparticleshaving an average zeta potential between +32 mV and +40 mV at a pH of 5.

In another preferred embodiment, the composition comprises nanoparticleshaving an average zeta potential between +5 mV and +25 mV at a pH of 5.

In another preferred embodiment, the composition comprises nanoparticleshaving an average zeta potential between +5 mV and +8 mV at a pH of 5.

In a preferred embodiment, the composition comprises nanoparticleshaving an average diameter less than 225 nm.

In another preferred embodiment, the composition comprises nanoparticleshaving an average diameter between 80 nm and 225 nm.

In another preferred embodiment, the composition comprises nanoparticleshaving an average diameter between 80 nm and 175 nm.

In a preferred embodiment, the composition has a DNA concentration ofbetween about 1 μg/ml and about 1.5 mg/ml, more preferably between about1 μg/ml and about 1 mg/ml, more preferably between about 10 μg/ml andabout 1 mg/ml, more preferably between about 50 μg/ml and about 1 mg/ml,more preferably between about 100 μg/ml and about 1 mg/ml, morepreferably between about 150 μg/ml and about 1 mg/ml, more preferablybetween about 200 μg/ml and about 1 mg/ml.

In a preferred embodiment, the composition has a pH of less than 6.5,more preferably less than 6.0, and most preferably between about 4.5 andabout 5.5.

In a preferred embodiment, the chitosan polymers of the nanoparticlehave a degree of deacetylation greater than about 70%, more preferablygreater than about 75%, more preferably greater than about 80%, morepreferably greater than about 85%, more preferably greater than about90%, more preferably greater than about 95%, and most preferably atleast 98%.

In a preferred embodiment, the composition comprises low molecularweight chitosan nanoparticles, wherein the plurality of chitosanpolymers of the low molecular weight nanoparticles have an averagemolecular weight between 3 kDa and 25 kDa. In a preferred embodiment,low molecular weight chitosan nanoparticles have an N:P ratio between10:1 and 90:1. In a preferred embodiment, low molecular weight chitosannanoparticles have an average zeta potential between +30 mV and +50 mV,more preferably between +30 mV and +40 mV at a pH of 5. In a preferredembodiment, low molecular weight chitosan nanoparticles have an averagediameter less than 175 nm.

In another embodiment, the composition comprises high molecular weightchitosan nanoparticles, wherein the plurality of chitosan polymers ofthe high molecular weight nanoparticles have an average molecular weightbetween 25 kDa and 250 kDa. In a preferred embodiment, high molecularweight chitosan nanoparticles have an N:P ratio between 2:1 and 40:1. Ina preferred embodiment, high molecular weight chitosan nanoparticleshave an average zeta potential between +5 mV and +25 mV at a pH of 5. Ina preferred embodiment, high molecular weight chitosan nanoparticleshave an average diameter less than 225 nm. In a preferred embodiment,high molecular weight chitosan nanoparticles have a chitosan:nucleicacid w/w ratio between 1:1 and 30:1.

In an especially preferred embodiment, the plurality of chitosanpolymers of the nanoparticle have an average molecular weight of about3.9 kDa, and a degree of deacetylation of about 98%. In a preferredembodiment, the composition comprises nanoparticles having an averagediameter less than 175 nm. In a preferred embodiment, the compositionhas a DNA concentration of between about 25 μg/ml and about 100 μg/ml.In a preferred embodiment, the nanoparticle has an N:P ratio of betweenabout 40:1 and about 80:1, most preferably an N:P ratio of about 60:1.In a preferred embodiment, the nanoparticle has a chitosan:nucleic acidw/w ratio between 20:1 and 40:1, most preferably a w/w ratio of 30:1.

In a further preferred embodiment, the plurality of chitosan polymers ofthe nanoparticle have an average molecular weight of about 3.9 kDa, anda degree of deacetylation of about 98%. In a preferred embodiment, thecomposition comprises nanoparticles having an average diameter of lessthan 175 nm. In a preferred embodiment, the composition has a DNAconcentration of between about 200 μg/ml and about 1 mg/ml. In apreferred embodiment, the nanoparticle has an N:P ratio of between about10:1 and about 30:1, most preferably an N:P ratio of about 20:1. In apreferred embodiment, the nanoparticle has a chitosan:nucleic acid w/wratio between 5:1 and 15:1, most preferably a w/w ratio of 10:1.

In a preferred embodiment, a nanoparticle of the invention is capable oftransfecting a gut mucosal precursor cell of the small intestine. In apreferred embodiment, the nanoparticle is capable of transfecting a gutmucosal precursor cell of the duodenum, jejunum, or ileum.

In a preferred embodiment, the nanoparticle is capable of transfecting agut mucosal precursor cell of the stomach.

In a preferred embodiment, the nanoparticle is capable of transfecting agut mucosal precursor cell of the colon.

In a preferred embodiment, the nanoparticle is capable of transfecting agut endocrine cell precursor cell. In a preferred embodiment, the gutendocrine cell precursor cell is capable of producing a gut endocrinecell selected from the group consisting of K cells, L-cells, S-cells,G-cells, D-cells, I-cells, Mo-cells, and Gr-cells. In an especiallypreferred embodiment, the gut endocrine cell precursor cell is a K cellprecursor cell.

In one embodiment, the nanoparticle lacks an endolysosomal peptide.

In one embodiment, the therapeutic nucleic acid of the nanoparticleencodes a therapeutic RNA. In a preferred embodiment, the therapeuticRNA is an siRNA, an antisense RNA, a short hairpin RNA, or an enzymaticRNA.

In a preferred embodiment, the therapeutic nucleic acid of thenanoparticle encodes a therapeutic protein. In an especially preferredembodiment, the therapeutic protein is a secreted therapeutic protein.Preferably, the nanoparticle is capable of increasing the systemic levelof the secreted therapeutic protein. Preferably, the nanoparticle iscapable of producing a long-term increase in the systemic level of thesecreted therapeutic protein. In one embodiment, the long-term increasein the systemic level of secreted therapeutic protein is static. In apreferred embodiment, the long-term increase in the systemic level ofsecreted therapeutic protein is dynamic.

In a preferred embodiment, the therapeutic nucleic acid of thenanoparticle encodes a therapeutic protein that is selected from thegroup consisting of hormones, enzymes, cytokines, chemokines,antibodies, mitogenic factors, growth factors, differentiation factors,factors influencing angiogenesis, factors influencing blood clotformation, factors influencing blood glucose levels, factors influencingglucose metabolism, factors influencing lipid metabolism, factorsinfluencing blood cholesterol levels, factors influencing blood LDL orHDL levels, factors influencing cell apoptosis, factors influencing foodintake, factors influencing energy expenditure, factors influencingappetite, factors influencing nutrient absorption, factors influencinginflammation, and factors influencing bone formation. Particularlypreferred are therapeutic nucleic acids encoding insulin, leptin,glucagon antagonist, GLP-1, GLP-2, Ghrelin, cholecystokinin, growthhormone, clotting factors, PYY, erythropoietin, inhibitors ofinflammation, IL-10, IL-17 antagonists, TNFα antagonists, growth hormonereleasing hormone, or parathyroid hormone. In a preferred embodiment,the encoded therapeutic protein is insulin. In another preferredembodiment, the encoded therapeutic protein is an insulin analog. Inanother preferred embodiment, the encoded therapeutic protein is leptin.In another preferred embodiment, the encoded therapeutic protein is PYY.

In a preferred embodiment, the therapeutic nucleic acid of thenanoparticle encodes a secreted therapeutic protein. In a preferredembodiment, the secreted therapeutic protein is capable of beingsecreted by regulated secretion from a gut endocrine cell. In apreferred embodiment, the secreted therapeutic protein is capable ofbeing secreted from a gut endocrine cell in response to a secretagogue,which is preferably a nutrient. In an especially preferred embodiment,the secreted therapeutic protein is capable of being secreted from a gutendocrine cell in response to glucose.

In one embodiment, a chitosan-based nanoparticle comprises two or moredistinct therapeutic nucleic acids.

In one embodiment, the expression control region of the nanoparticledoes not comprise a CMV promoter.

In one embodiment, the expression control region of the nanoparticledoes not comprise a viral promoter.

In a preferred embodiment, the expression control region of thenanoparticle comprises a non-constitutive promoter.

In a preferred embodiment, the expression control region of thenanoparticle comprises a gut-specific control sequence.

In a preferred embodiment, the expression control region of thenanoparticle comprises a mucosal cell-specific control sequence.

In a preferred embodiment, the expression control region of thenanoparticle comprises a gut endocrine cell-specific control sequence.

In a preferred embodiment, the expression control region of thenanoparticle comprises an inducible promoter. In one embodiment, thepromoter is regulatable by a small molecule chemical compound. Inanother embodiment, the promoter is a nutrient-regulatable promoter. Inone embodiment, the nutrient-regulatable promoter is regulated byglucose. In an especially preferred embodiment, the nutrient-regulatablepromoter is a GIP promoter.

In a preferred embodiment, the therapeutic construct of the nanoparticlefurther comprises an integration sequence. In one embodiment, thetherapeutic construct comprises a single integration sequence. Inanother embodiment, the therapeutic construct comprises a first and asecond integration sequence, which first and second integrationsequences flank the expression control region operably linked to thetherapeutic nucleic acid (i.e., the expression control region andtherapeutic nucleic acid taken together). In a preferred embodiment, theintegration sequence(s) is functional in combination with a means forintegration that is selected from the group consisting of mariner,sleeping beauty, FLP, Cre, φC31, R, lambda, and means for integrationfrom integrating viruses such as AAV, retroviruses, and lentiviruses.

In a preferred embodiment, a chitosan-based nanoparticle furthercomprises a non-therapeutic construct in addition to a therapeuticconstruct, wherein the non-therapeutic construct comprises a nucleicacid sequence encoding a means for integration operably linked to asecond expression control region that is functional in a gut mucosalprecursor cell. This second expression control region and the expressioncontrol region operably linked to the therapeutic nucleic acid may bethe same or different. The encoded means for integration is preferablyselected from the group consisting of mariner, sleeping beauty, FLP,Cre, φC31, R, lambda, and means for integration from integrating virusessuch as AAV, retroviruses, and lentiviruses.

In one embodiment, the nanoparticle consists essentially of (i) aplurality of chitosan polymers having an average molecular weightbetween 3 kDa and 250 kDa, (ii) a therapeutic construct, wherein thetherapeutic construct comprises a therapeutic nucleic acid operablylinked to an expression control region that is functional in a gutmucosal cell, wherein the therapeutic nucleic acid is capable ofexerting a therapeutic effect when expressed in the gut mucosal cell,and (iii) a non-therapeutic construct, wherein said non-therapeuticconstruct comprises a nucleic acid sequence encoding a means forintegration operably linked to a second expression control region thatis functional in a gut mucosal precursor cell.

In one aspect, the invention provides a chitosan-based nanoparticlecapable of transfecting a gut mucosal precursor cell in vivo, comprising(i) a plurality of chitosan polymers, and (ii) a non-therapeuticconstruct, wherein the non-therapeutic construct comprises a nucleicacid encoding a means for integration operably linked to an expressioncontrol region that is functional in a gut mucosal precursor cell. Theencoded means for integration is preferably selected from the groupconsisting of mariner, sleeping beauty, FLP, Cre, φC31, R, lambda, andmeans for integration from integrating viruses such as AAV,retroviruses, and lentiviruses.

In one embodiment, the nanoparticle consists essentially of (i) aplurality of chitosan polymers, and (ii) a non-therapeutic construct,wherein the non-therapeutic construct comprises a nucleic acid encodinga means for integration operably linked to an expression control regionthat is functional in a gut mucosal precursor cell.

In one aspect, the invention provides a method for transfecting a gutmucosal cell with a therapeutic nucleic acid in vivo, comprisingcontacting gut mucosa in vivo with a chitosan-based nanoparticle of theinvention.

In a preferred embodiment, the method involves contacting mucosa of thesmall intestine. In a preferred embodiment, the method involvescontacting mucosa of the duodenum, jejunum, or ileum with achitosan-based nanoparticle of the invention.

In a preferred embodiment, the method involves contacting mucosa of thestomach with a chitosan-based nanoparticle of the invention.

In a preferred embodiment, the method involves contacting mucosa of thecolon with a chitosan-based nanoparticle of the invention.

In a preferred embodiment, the chitosan-based nanoparticle transfects agut mucosal precursor cell in the gut mucosa. In a preferred embodiment,the gut mucosal precursor cell is a gut endocrine cell precursor cell.In a preferred embodiment, the gut endocrine cell precursor cellproduces a gut endocrine cell selected from the group consisting of Kcells, L-cells, S-cells, G-cells, D-cells, I-cells, Mo-cells, andGr-cells. In an especially preferred embodiment, the gut endocrine cellprecursor cell produces a K cell.

In a preferred embodiment, the gut mucosal precursor cell is a mucosalcell of the small intestine. In a preferred embodiment, the gut mucosalprecursor cell is a mucosal cell of the duodenum, jejunum, or ileum.

In a preferred embodiment, the gut mucosal precursor cell is a mucosalcell of the stomach.

In a preferred embodiment, the gut mucosal precursor cell is a mucosalcell of the colon.

In a preferred embodiment, the gut mucosal precursor cell produces amucosal cell that expresses the therapeutic nucleic acid.

In one embodiment, the therapeutic nucleic acid encodes a therapeuticRNA.

In a preferred embodiment, the therapeutic nucleic acid encodes atherapeutic protein. In a preferred embodiment, the therapeutic proteinis a secreted therapeutic protein.

In a preferred embodiment, the therapeutic nucleic acid of thenanoparticle encodes a therapeutic protein that is selected from thegroup consisting of hormones, enzymes, cytokines, chemokines,antibodies, mitogenic factors, growth factors, differentiation factors,factors influencing angiogenesis, factors influencing blood clotformation, factors influencing blood glucose levels, factors influencingglucose metabolism, factors influencing lipid metabolism, factorsinfluencing blood cholesterol levels, factors influencing blood LDL orHDL levels, factors influencing cell apoptosis, factors influencing foodintake, factors influencing energy expenditure, factors influencingappetite, factors influencing nutrient absorption, factors influencinginflammation, and factors influencing bone formation. Particularlypreferred are therapeutic nucleic acids encoding insulin, leptin,glucagon antagonist, GLP-1, GLP-2, Ghrelin, cholecystokinin, growthhormone, clotting factors, PYY, erythropoietin, inhibitors ofinflammation, IL-10, IL-17 antagonists, TNFα antagonists, growth hormonereleasing hormone, or parathyroid hormone. In a preferred embodiment,the encoded therapeutic protein is insulin. In another preferredembodiment, the encoded therapeutic protein is an insulin analog. Inanother preferred embodiment, the encoded therapeutic protein is leptin.In another preferred embodiment, the encoded therapeutic protein is PYY.

In a preferred embodiment, the therapeutic protein is produced in a gutmucosal cell and enters the systemic circulation such that the systemiclevel of the therapeutic protein is increased. In a preferredembodiment, the therapeutic protein is released by regulated secretioninto the systemic circulation.

In a preferred embodiment, the systemic level of the therapeutic proteinis increased for longer than about 4 days, more preferably longer thanabout 5 days, more preferably longer than about 6 days, more preferablylonger than about 7 days, more preferably longer than about 10 days,more preferably longer than about 2 weeks, more preferably longer thanabout 3 weeks, more preferably longer than about 4 weeks, morepreferably longer than about 6 weeks, more preferably longer than about8 weeks, more preferably longer than about 10 weeks, and most preferablylonger than about 12 weeks.

In one embodiment, the increase in the systemic level of the therapeuticprotein is static. In a preferred embodiment, the increase in thesystemic level of the therapeutic protein is dynamic.

In one embodiment, the method comprises contacting gut mucosa in vivowith a first chitosan-based nanoparticle and a second chitosan-basednanoparticle. The first chitosan based nanoparticle is capable oftransfecting a gut mucosal precursor cell in vivo and comprises (i) aplurality of chitosan polymers, and (ii) a therapeutic construct,wherein the therapeutic construct comprises a therapeutic nucleic acidoperably linked to an expression control region functional in a gutmucosal cell, and an integration sequence. The second chitosan-basednanoparticle is capable of transfecting a gut mucosal precursor cell invivo and comprises (i) a plurality of chitosan polymers, and (ii) anon-therapeutic construct, wherein the non-therapeutic constructcomprises a nucleic acid encoding a means for integration operablylinked to an expression control region that is functional in a gutmucosal precursor cell. Without being bound by theory, it appears that agut mucosal precursor cell in the gut mucosa is transfected with thefirst and second nanoparticles. The nucleic acid of the secondnanoparticle is expressed in the gut mucosal precursor cell to produce ameans for integration, whereby the means for integration integrates thetherapeutic nucleic acid operably linked to an expression control regionprovided by the first nanoparticle into the genome of the gut mucosalprecursor cell.

In another embodiment, the method comprises contacting gut mucosa invivo with a chitosan-based nanoparticle comprising (i) a plurality ofchitosan polymers; (ii) a therapeutic construct, wherein the therapeuticconstruct comprises a therapeutic nucleic acid operably linked to anexpression control region functional in a gut mucosal cell, and anintegration sequence; and (iii) a non-therapeutic construct, wherein thenon-therapeutic construct comprises a nucleic acid encoding a means forintegration operably linked to an expression control region that isfunctional in a gut mucosal precursor cell. Without being bound bytheory, it appears that a gut mucosal precursor cell in the gut mucosais transfected using the nanoparticle. The nucleic acid encoding a meansfor integration is expressed in the gut mucosal precursor cell toproduce a means for integration, whereby the means for integrationintegrates the therapeutic nucleic acid operably linked to an expressioncontrol region into the genome of the gut mucosal precursor cell.

It will be appreciated that optimal ratios of therapeutic andnon-therapeutic constructs for facilitating integration may varydepending upon the particular means of integration contemplated for use.Determining the optimal ratios of therapeutic to non-therapeuticconstructs is done by one of reasonable skill in the art without undueexperimentation.

In one aspect, the invention provides methods for effecting long-termexpression of a therapeutic nucleic acid in a mammalian gut mucosalcell. The methods involve contacting the gut mucosa of a mammal with achitosan-based nanoparticle of the invention, wherein the nanoparticlecomprises a therapeutic nucleic acid, and wherein the therapeuticnucleic acid is expressed in a gut mucosal cell of the mammal.

In one embodiment, the methods comprise the use of a nanoparticle of theinvention, which nanoparticle comprises a therapeutic construct and anon-therapeutic construct.

In one embodiment, the methods comprise the use of two nanoparticles ofthe invention, the first comprising a therapeutic construct and thesecond comprising a non-therapeutic construct.

In one aspect, the invention provides methods for increasing thesystemic level of a secreted therapeutic protein in a mammal. Themethods comprise contacting gut mucosa of a mammal with a chitosan-basednanoparticle of the invention, wherein the nanoparticle comprises atherapeutic nucleic acid encoding a secreted therapeutic protein,wherein the secreted therapeutic protein is produced in a mucosal cellof the gut mucosa, and wherein the secreted therapeutic protein producedin the mucosal cell enters the systemic circulation such that thesystemic level of the secreted therapeutic protein is increased.

In one embodiment, the methods comprise the use of a nanoparticle of theinvention, which nanoparticle comprises a therapeutic construct and anon-therapeutic construct.

In one embodiment, the methods comprise the use of two nanoparticles ofthe invention, the first comprising a therapeutic construct and thesecond comprising a non-therapeutic construct.

In one aspect, the invention provides methods for treating patientshaving diseases or conditions treatable by increasing the systemic levelof a therapeutic protein. The methods comprise contacting gut mucosa ofa patient with a chitosan-based nanoparticle of the invention, whereinthe nanoparticle comprises a therapeutic nucleic acid encoding asecreted therapeutic protein, wherein the secreted therapeutic proteinis produced in a mucosal cell of the gut mucosa of the patient, andwherein the secreted therapeutic protein produced in the mucosal cellenters the systemic circulation such that the systemic level of thesecreted therapeutic protein is increased to a therapeutically effectivelevel.

In a preferred embodiment, the disease is a metabolic disease.

In a preferred embodiment, the disease is diabetes mellitus.

In another preferred embodiment, the condition is morbid obesity.

In another preferred embodiment, the condition is growth deficiency.

In a preferred embodiment, the chitosan-based nanoparticle is orallyadministered.

In a preferred embodiment, the chitosan-based nanoparticle isadministered endoscopically.

In a preferred embodiment, the chitosan-based nanoparticle isadministered rectally.

In a preferred embodiment, the mucosal cell is a gut endocrine cell. Ina preferred embodiment, the gut endocrine cell is selected from thegroup consisting of K cells, L-cells, S-cells, G-cells, D-cells,I-cells, Mo-cells, and Gr-cells. In an especially preferred embodiment,the gut endocrine cell is a K cell.

In a preferred embodiment, the mucosal cell is a mucosal cell of thesmall intestine. In a preferred embodiment, the mucosal cell is amucosal cell of the duodenum, jejunum, or ileum.

In a preferred embodiment, the mucosal cell is a mucosal cell of thestomach.

In a preferred embodiment, the mucosal cell is a mucosal cell of thecolon.

In a preferred embodiment, the therapeutic nucleic acid of thenanoparticle encodes a therapeutic protein that is selected from thegroup consisting of hormones, enzymes, cytokines, chemokines,antibodies, mitogenic factors, growth factors, differentiation factors,factors influencing angiogenesis, factors influencing blood clotformation, factors influencing blood glucose levels, factors influencingglucose metabolism, factors influencing lipid metabolism, factorsinfluencing blood cholesterol levels, factors influencing blood LDL orHDL levels, factors influencing cell apoptosis, factors influencing foodintake, factors influencing energy expenditure, factors influencingappetite, factors influencing nutrient absorption, factors influencinginflammation, and factors influencing bone formation. Particularlypreferred are therapeutic nucleic acids encoding insulin, leptin,glucagon antagonist, GLP-1, GLP-2, Ghrelin, cholecystokinin, growthhormone, clotting factors, PYY, erythropoietin, inhibitors ofinflammation, IL-10, IL-17 antagonists, TNFα antagonists, growth hormonereleasing hormone, or parathyroid hormone. In a preferred embodiment,the encoded therapeutic protein is insulin. In another preferredembodiment, the encoded therapeutic protein is an insulin analog. Inanother preferred embodiment, the encoded therapeutic protein is leptin.In another preferred embodiment, the encoded therapeutic protein is PYY.

In a preferred embodiment, the secreted therapeutic protein is releasedby regulated secretion from a gut endocrine cell.

In a preferred embodiment, the systemic level of the secretedtherapeutic protein is increased for longer than about 4 days, morepreferably longer than about 5 days, more preferably longer than about 6days, more preferably longer than about 7 days, more preferably longerthan about 10 days, more preferably longer than about 2 weeks, morepreferably longer than about 3 weeks, more preferably longer than about4 weeks, more preferably longer than about 6 weeks, more preferablylonger than about 8 weeks, more preferably longer than about 10 weeks,and most preferably longer than about 12 weeks.

In one embodiment, the methods comprise the use of a nanoparticle of theinvention, which nanoparticle comprises a therapeutic construct and anon-therapeutic construct.

In one embodiment, the methods comprise the use of two nanoparticles ofthe invention, the first comprising a therapeutic construct and thesecond comprising a non-therapeutic construct.

In one aspect, the invention provides pharmaceutical compositionscomprising chitosan-based nanoparticles of the invention.

In one embodiment, the invention provides pharmaceutical compositionscapable of increasing the systemic level of therapeutic proteins. Such apharmaceutical composition comprises a chitosan-based nanoparticle ofthe invention, wherein the nanoparticle comprises a therapeutic nucleicacid encoding a secreted therapeutic protein.

In one embodiment, the pharmaceutical composition comprises two or moredistinct therapeutic nucleic acids of the invention.

In one embodiment, the pharmaceutical composition comprises a firstchitosan-based nanoparticle of the invention and a second chitosan-basednanoparticle of the invention, wherein the first and second nanoparticlecomprise distinct therapeutic nucleic acids.

In one embodiment, the pharmaceutical composition comprises atherapeutic construct and a non-therapeutic construct of the invention.

In one embodiment, the pharmaceutical composition comprises a firstchitosan-based nanoparticle of the invention and a second chitosan-basednanoparticle of the invention, wherein the first nanoparticle comprisesa therapeutic construct, and the second nanoparticle comprises anon-therapeutic construct.

In a preferred embodiment, the pharmaceutical composition is capable ofincreasing the systemic level of the therapeutic protein for longer thanabout 4 days, more preferably longer than about 5 days, more preferablylonger than about 6 days, more preferably longer than about 7 days, morepreferably longer than about 10 days, more preferably longer than about2 weeks, more preferably longer than about 3 weeks, more preferablylonger than about 4 weeks, more preferably longer than about 6 weeks,more preferably longer than about 8 weeks, more preferably longer thanabout 10 weeks, and most preferably longer than about 12 weeks.

In a preferred embodiment, the pharmaceutical composition may beadministered orally.

In a preferred embodiment, the pharmaceutical composition may beadministered endoscopically.

In a preferred embodiment, the pharmaceutical composition may beadministered rectally.

In one aspect, the invention provides a modified gut mucosal cell, whichis produced by contacting gut mucosa in vivo with a chitosan-basednanoparticle according to a method disclosed herein.

In one aspect, the invention provides methods of producingchitosan-based nanoparticles of the invention. In one embodiment,methods for producing nanoparticles capable of producing long-termexpression of therapeutic nucleic acids in gut mucosal cells areprovided. The methods involve the formation of a nanoparticlepreparation mixture.

In a preferred embodiment, chitosan polymers having an average molecularweight from about 3 kDa to about 250 kDa are used.

In another preferred embodiment, chitosan polymers having an averagemolecular weight from about 3 kDa to about 210 kDa are used.

In another preferred embodiment, chitosan polymers having an averagemolecular weight from about 10 kDa to about 250 kDa are used.

In another preferred embodiment, chitosan polymers having an averagemolecular weight from about 10 kDa to about 210 kDa are used.

In another preferred embodiment, chitosan polymers having an averagemolecular weight from about 3 kDa to about 50 kDa are used.

In another preferred embodiment, chitosan polymers having an averagemolecular weight from about 3 kDa to about 6 kDa are used.

In another preferred embodiment, chitosan polymers having an averagemolecular weight from about 200 kDa to about 210 kDa are used.

In a preferred embodiment, the nanoparticle preparation mixture has anamine:phosphate (N:P) ratio from about 1:1 to about 100:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P ratio from about 1:1 to about 6:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P ratio from about 1:1 to about 4:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P of about 3:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P ratio from about 10:1 to about 90:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P ratio from about 10:1 to about 50:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P ratio from about 10:1 to about 40:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P ratio from about 10:1 to about 30:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P ratio from about 60:1 to about 100:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P ratio from about 70:1 to about 100:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P ratio of about 20:1.

In another preferred embodiment, the nanoparticle preparation mixturehas an N:P ratio of about 60:1.

In a preferred embodiment, the nanoparticle preparation mixture has achitosan:nucleic acid w/w ratio from about 1:1 to about 50:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio from about 1:1 to about 5:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio from about 1:1 to about 3:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio of about 2:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio from about 5:1 to about 45:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio from about 5:1 to about 25:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio from about 5:1 to about 20:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio from about 5:1 to about 15:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio from about 30:1 to about 50:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio from about 35:1 to about 50:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio of about 10:1.

In another preferred embodiment, the nanoparticle preparation mixturehas a chitosan:nucleic acid w/w ratio of about 30:1.

In one aspect, the invention provides chitosan-based nanoparticlesproduced by a nanoparticle production method disclosed herein.

In one aspect, the invention provides a method for preparing amedicament useful for the treatment of a disease or condition that istreatable by increasing the systemic level of a secreted therapeuticprotein. The methods involve methods of producing chitosan-basednanoparticles disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of in vitro transfection of 293 T cells withchitosan-based nanoparticles comprising chitosan polymers of variousmolecular weights, as indicated.

FIG. 2 shows results of in vivo transfection of murine gut mucosal cellsof the duodenum with chitosan-based nanoparticles comprising chitosanpolymers of various molecular weights, as indicated.

FIG. 3 shows results of in vivo transfection of murine gut mucosal cellsof the duodenum with chitosan-based nanoparticles comprising chitosanpolymers of various molecular weights and at various N:P ratios, asindicated.

FIG. 4 shows results of in vivo transfection of murine gut mucosal cellsof the duodenum with RCO5 chitosan-based nanoparticles at an N:P ratioof 2.85:1, compared to transduction with FIV and AAV particles.

FIG. 5 shows results of in vivo transfection (single administration) ofmurine gut mucosal cells of the duodenum with P1 chitosan-basednanoparticles at an N:P ratio of 60:1, wherein the nanoparticlecomprises an EF1α-SEAP construct. The level of SEAP protein in blood atvarious time points is shown.

FIG. 6 shows results of in vivo transfection (single administration) ofmurine gut mucosal cells of the duodenum with P1 chitosan-basednanoparticles at an N:P ratio of 60:1 wherein the nanoparticles comprise(i) CMV-lacZ integration construct (pMM2611-beta-gal), and (ii)CMV-Mariner expression construct (pCMV-C9.gck). Gene copy number at 14days post transfection is shown.

FIG. 7A shows schematic of CMV-lacZ integration construct(pMM2611-beta-gal) used with Mariner transposase. FIG. 7B showsschematic of Mariner expression construct (pCMV-C9.gck).

FIG. 8 shows results of chitosan mediated gene transfer to duodenalmucosal cells in mice at 2 days post delivery, and persistence at 14days post delivery with use of φC31.

FIG. 9 shows results of single administration of χφ-GEMS™ carrying theGIP-hlNS gene to achieve long-term systemic production of human insulinin mice.

FIG. 10 shows results of glucose and meal challenge in mice treated withchitosan-based nanoparticle carrying GIP-insulin gene.

FIG. 11 shows results of a matrix study analyzing the production ofhuman C-peptide over time in mice treated with different chitosan-basednanoparticles carrying GIP-hlNS gene.

FIG. 12 shows results of human insulin mRNA measurement in duodenum ofpigs treated with chitosan-based nanoparticles carrying hGIP-hlNS geneat 7 days post delivery.

FIG. 13 shows results of a matrix study analyzing the production ofhuman insulin in pigs treated with different chitosan-basednanoparticles carrying hGIP-hlNS gene.

FIG. 14 shows transformation of stomach mucosal cells by oral deliveryof a chitosan-packaged DNA polyplex. Levels of insulin and SEAP geneexpression levels in the stomach mucosa of mice at 2 days after oraladministration of vector. NTC=non-treated controls, T=treated animals.

DETAILED DESCRIPTION

Gut Mucosal Cells

As used herein, “gut mucosal cell” refers to a cell of the gut mucosa.Included among gut mucosal cells are endocrine cells, non-endocrinecells, and precursors thereof. Gut mucosal precursor cells include stemcells. A gut mucosal precursor cell is directly or indirectly aprecursor of a differentiated cell type of the gut mucosa. Gut mucosalprecursor cells include undifferentiated gut mucosal cells. Gut mucosalprecursor cells give rise to major cell types of the gut mucosa,including endocrine cells and non-endocrine cells. A gut endocrine cellprecursor cell is a precursor of a gut mucosal cell that is a gutendocrine cell, e.g., a K cell.

Particular examples of gut mucosal cells include endocrine cells, suchas K cells, L-cells, S-cells, G-cells, D-cells, I-cells, Mo-cells,Gr-cells, and non-endocrine cells such as absorptive enterocytes.Endocrine cells are generally characterized by their ability to secretea synthesized protein into the blood in response to a signal or stimuli(a “secretagogue”). Non-endocrine cells are generally not known tosecrete a synthesized protein into the blood in response to a signal orstimuli.

Especially preferred gut endocrine cells for use in the invention are Kcells (Sandstrom O., El-Salhy M., Mech. Ageing Dev. 108:39 (1999)).

A partial list of several types of gut endocrine cells and proteinsnormally produced thereby are shown in Table 1.

TABLE 1 Cell Type Peptide G-cells Gastrin D-cells Somatostatin K-cellsGlucose-dependent Insulinotropic Polypeptide L-cells GLP-1 GLP-2 I-cellsCholycystokinin Mo-cells Motilin Gr-cells Ghrelin

Chitosan-Based Nanoparticles

By “chitosan-based nanoparticle”, or “DNA/chitosan particle” is meant acomplex comprising a plurality of chitosan polymers and a DNA molecule.“Polyplex” is used interchangeably with “chitosan-based nanoparticle”herein. The chitosan polymers of chitosan-based nanoparticles preferablyhave an average molecular weight of less than about 250 kDa. The DNAmolecule in the context of the chitosan-based nanoparticle isdeliverable to a cell in vivo. Frequently, a chitosan-based nanoparticleis referred to herein as a nanoparticle.

As used herein, average weight of chitosan polymers refers to the weightaverage molecular weight.

In one embodiment, chitosan is obtained by the method of Richardson etal., Int. J. Pharmaceutics, 178:231-243, 1999.

In one embodiment, high molecular weight chitosan-based nanoparticlesare produced as follows. Plasmid DNA and chitosan solutions, preparedseparately, are adjusted to a concentration equal to two-times therequired final concentration. DNA is diluted in water or 50 mM sodiumsulfate solution. A desired molecular weight chitosan polymerpreparation, which comprises chitosan polymers preferably having anaverage molecular weight less than 250 kDa, is dissolved in 5 mM sodiumacetate, pH 5.5. Both solutions are incubated at 55° C. for 5 minutesbefore being combined to form a chitosan-based nanoparticle preparationmixture. Equal volumes of the two solutions are mixed and rapidlyvortexed for 30 seconds to form DNA/chitosan particles. This preparationmay be further diluted in various buffers prior to use. Production ofthe nanoparticles of the invention does not require lyophilization anduse of lyophilized product in an acetylation reaction.

In a preferred embodiment, chitosan-based nanoparticles, especially lowmolecular weight chitosan-based nanoparticles, are produced as follows.Chitosan powder is added to 0.5% aqueous solution of acetic acid untilthe chitosan working solution reaches pH 4.8. The working solution isthen filtered through a membrane filter (Acrodisc 0.2 μm pore size, PallLife Sciences). Stock DNA solutions (in 1×TE) of plasmid A and plasmid Bare mixed in a 5:1 ratio of plasmid A to plasmid B, diluted in water,and then filtered through a membrane filter (Acrodisc 0.2 μm pore size,Pall Life Sciences) to produce the DNA working solution. See theexperimental section for further details.

The chitosan polymers of chitosan-based nanoparticles preferably have anaverage molecular weight of less than about 250 kDa, more preferablyless than 230 kDa, more preferably less than 220 kDa. In a preferredembodiment, the plurality of chitosan polymers of the nanoparticle havean average molecular weight from about 3 kDa to about 250 kDa. Otherranges of average molecular weight for chitosan polymers in preferrednanoparticles of the invention include 3-6 kDa, 3-10 kDa, 3-50 kDa,3-210 kDa, 10-210 kDa, 10-250 kDa, and 210-250 kDa.

A preferred DNA concentration in the chitosan-based nanoparticlepreparation mixture is in the range of about 1 μg/ml to about 1.5 mg/ml,more preferably between about 10 μg/ml to about 1 mg/ml, more preferablybetween about 25 μg/ml to about 1 mg/ml, more preferably between about50 μg/ml to about 1 mg/ml, more preferably between about 100 μg/ml toabout 1 mg/ml.

A preferred chitosan concentration in the chitosan-based nanoparticlepreparation mixture is in the range of 0.001% to 1.0%, w/w.

In a preferred embodiment, the chitosan-based nanoparticle has anamine:phosphate (N:P) ratio from about 1:1 to about 100:1. Other N:Pratio ranges of preferred chitosan-based nanoparticles of the inventioninclude about 1:1 to about 6:1, about 1:1 to about 4:1, about 10:1 toabout 90:1, about 10:1 to about 50:1, about 10:1 to about 40:1, about10:1 to about 30:1, about 60:1 to about 100:1, and about 70:1 to about100:1.

The amine content of chitosan may be varied by varying the degree ofchitosan acetylation. The chitosan polymers used in the chitosan-basednanoparticles of the invention preferably have between 70% and 100%,more preferably between 80% and 100%, more preferably between 90% and100% deacetylation.

In a preferred embodiment, the chitosan-based nanoparticle has achitosan:nucleic acid w/w ratio from about 1:1 to about 50:1. Otherchitosan:nucleic acid w/w ratio ranges of preferred chitosan-basednanoparticles include about 1:1 to about 5:1, about 1:1 to about 3:1,about 5:1 to about 45:1, about 5:1 to about 25:1, about 5:1 to about20:1, about 5:1 to about 15:1, about 30:1 to about 50:1, and about 35:1to about 50:1.

In a preferred embodiment, the composition comprises nanoparticleshaving an average zeta potential between +5 mV and +50 mV at a pH of 5.

In another preferred preferred embodiment, the composition comprisesnanoparticles having an average zeta potential between +30 mV and +50 mVat a pH of 5.

In another preferred preferred embodiment, the composition comprisesnanoparticles having an average zeta potential between +30 mV and +40 mVat a pH of 5.

In another preferred embodiment, the composition comprises nanoparticleshaving an average zeta potential between +32 mV and +40 mV at a pH of 5.

In another preferred embodiment, the composition comprises nanoparticleshaving an average zeta potential between +5 mV and +25 mV at a pH of 5.

In another preferred embodiment, the composition comprises nanoparticleshaving an average zeta potential between +5 mV and +8 mV at a pH of 5.

In a preferred embodiment, the composition comprises nanoparticleshaving an average diameter less than 225 nm.

In another preferred embodiment, the composition comprises nanoparticleshaving an average diameter between 80 nm and 225 nm.

In another preferred embodiment, the composition comprises nanoparticleshaving an average diameter between 80 nm and 175 nm.

In a preferred embodiment, the composition has a DNA concentration ofbetween about 1 μg/ml and about 1.5 mg/ml, more preferably between about1 μg/ml and about 1 mg/ml, more preferably between about 10 μg/ml andabout 1 mg/ml, more preferably between about 50 μg/ml and about 1 mg/ml,more preferably between about 100 μg/ml and about 1 mg/ml, morepreferably between about 150 μg/ml and about 1 mg/ml, more preferablybetween about 200 μg/ml and about 1 mg/ml.

In a preferred embodiment, the composition has a pH of less than 6.5,more preferably less than 6.0, and most preferably between about 4.5 andabout 5.5.

In a preferred embodiment, the chitosan polymers of the nanoparticlehave a degree of deacetylation greater than about 70%, more preferablygreater than about 75%, more preferably greater than about 80%, morepreferably greater than about 85%, more preferably greater than about90%, more preferably greater than about 95%, and most preferably atleast 98%.

In a preferred embodiment, the composition comprises low molecularweight chitosan nanoparticles, wherein the plurality of chitosanpolymers of the low molecular weight nanoparticles have an averagemolecular weight between 3 kDa and 25 kDa. In a preferred embodiment,low molecular weight chitosan nanoparticles have an N:P ratio between10:1 and 90:1. In a preferred embodiment, low molecular weight chitosannanoparticles have an average zeta potential between +30 mV and +50 mV,more preferably between +30 mV and +40 mV at a pH of 5. In a preferredembodiment, low molecular weight chitosan nanoparticles have an averagediameter less than 175 nm.

In another embodiment, the composition comprises high molecular weightchitosan nanoparticles, wherein the plurality of chitosan polymers ofthe high molecular weight nanoparticles have an average molecular weightbetween 25 kDa and 250 kDa. In a preferred embodiment, high molecularweight chitosan nanoparticles have an N:P ratio between 2:1 and 40:1. Ina preferred embodiment, high molecular weight chitosan nanoparticleshave an average zeta potential between +5 mV and +25 mV at a pH of 5. Ina preferred embodiment, high molecular weight chitosan nanoparticleshave an average diameter less than 225 nm. In a preferred embodiment,high molecular weight chitosan nanoparticles have a chitosan:nucleicacid w/w ratio between 1:1 and 30:1.

In an especially preferred embodiment, the plurality of chitosanpolymers of the nanoparticle have an average molecular weight of about3.9 kDa, and a degree of deacetylation of about 98%. In a preferredembodiment, the composition comprises nanoparticles having an averagediameter less than 175 nm. In a preferred embodiment, the compositionhas a DNA concentration of between about 25 μg/ml and about 100 μg/ml.In a preferred embodiment, the nanoparticle has an N:P ratio of betweenabout 40:1 and about 80:1, most preferably an N:P ratio of about 60:1.In a preferred embodiment, the nanoparticle has a chitosan:nucleic acidw/w ratio between 20:1 and 40:1, most preferably a w/w ratio of 30:1.

In a further preferred embodiment, the plurality of chitosan polymers ofthe nanoparticle have an average molecular weight of about 3.9 kDa, anda degree of deacetylation of about 98%. In a preferred embodiment, thecomposition comprises nanoparticles having an average diameter of lessthan 175 nm. In a preferred embodiment, the composition has a DNAconcentration of between about 200 μg/ml and about 1 mg/ml. In apreferred embodiment, the nanoparticle has an N:P ratio of between about10:1 and about 30:1, most preferably an N:P ratio of about 20:1. In apreferred embodiment, the nanoparticle has a chitosan:nucleic acid w/wratio between 5:1 and 15:1, most preferably a w/w ratio of 10:1.

Procedures or features that may be used or added to alter transfectionefficiency include co-complexation of lysosomolytic agents innanoparticles, though lysosomolytic agents are not required. Combinationnanoparticles that include additional therapeutic agents co-complexedare contemplated. These additional agents and lysosomolytic agents maybe co-complexed during complex formation.

Additionally, the stability of nanoparticles may be varied bycrosslinking.

Additionally, ligands or targeting moieties may be conjugated orotherwise affixed to nanoparticles to enhance specificity or uptake, forexample, by receptor-mediated endocytosis.

In a number of preferred embodiments, a chitosan-based nanoparticle ofthe invention is capable of producing long-term expression of atherapeutic nucleic acid in a gut mucosal cell. Differentiated gutmucosal cells are typically short-lived. As used herein, long-termexpression in a mucosal cell refers to expression in one or more mucosalcells, and expression in the mucosa is preferably for more than about 4days. Without being bound by theory, in the context of the smallintestine, which is a preferred location for mucosal cell transfectionin the present invention, a nanoparticle of the invention may enter acrypt of the gut mucosa and transfect a gut mucosal precursor cell. Thisprovides for long-term expression of a therapeutic nucleic acid relativeto transfection of a terminally differentiated and shorter-lived mucosalcell. Additionally, in embodiments wherein a therapeutic constructcomprises an integration sequence, the nanoparticle provides for genomicintegration of the therapeutic nucleic acid into the genome of aprecursor cell (including stem cells) and, in a preferred embodiment,long-term expression of a therapeutic nucleic acid in a series ofdifferentiated mucosal cells arising from the precursor cell.Accordingly, as used herein, long-term expression in a mucosal cell doesnot necessarily refer to expression in the same cell for the duration.

In some embodiments, the expression control region of a therapeuticconstruct possesses constitutive activity, providing for the staticexpression of a therapeutic nucleic acid. In a number of preferredembodiments, the expression control region of a therapeutic constructdoes not have constitutive activity. This provides for the dynamicexpression of a therapeutic nucleic acid. By “dynamic” expression ismeant expression that changes over time. Such expression over time caninclude a period of no expression or undetectable expression, duringwhich a therapeutic nucleic acid is present in a mucosal cell but is notexpressed or is not expressed at a detectable level. Dynamic expressionmay include several such periods of low or absent expression separatedby periods of detectable expression. In a number of preferredembodiments, the therapeutic nucleic acid is operably linked to aregulatable promoter. This provides for the regulatable expression oftherapeutic nucleic acids.

The ability to produce long-term expression of a therapeutic nucleicacid is an identifying characteristic of many preferred chitosan-basednanoparticles of the invention. The ability to produce long-termexpression refers to the ability of a preferred nanoparticle to producelong-term expression after a single administration, although somemethods herein involve repeated administrations of chitosan-basednanoparticles. Notably, in embodiments wherein a nanoparticle is capableof producing long-term expression of a therapeutic nucleic acid, thelong-term expression of such a therapeutic nucleic acid may be dynamic,with periods of low or undetectable expression.

Expression Control Regions

Expression control regions comprise regulatory polynucleotides(sometimes referred to herein as elements), such as promoters andenhancers, that influence expression of an operably linked therapeuticnucleic acid. Preferred expression control regions are those that areselectively active in gut tissue or a particular gut tissue cell type.

In a number of preferred embodiments, an expression control region of atherapeutic construct comprises a gut-specific promoter. A gut-specificpromoter exhibits activity in gut mucosal cells and potentially othergut cells. In a preferred embodiment, a gut-specific promoter does notexhibit substantial activity in a wide variety of other tissues. Viralpromoters such as the CMV promoter which exhibit activity in a widevariety of cell types are not gut-specific promoters. Gut specificpromoters may exhibit constitutive or non-constitutive activity.Especially preferred are regulatable gut-specific promoters.

For example, the promoter of the proglucagon gene comprises gut-specificelements and finds use in the present invention (Lee, Y. C., et al. J.Biol. Chem. 267:10705 (1992); Gajic and Drucker, Endocrinol. 132:1055(1993).

In a number of preferred embodiments, an expression control region of atherapeutic construct comprises a gut mucosal cell-specific promoter. Agut mucosal cell-specific promoter exhibits activity in gut mucosalcells. In a preferred embodiment, a gut mucosal cell-specific promoterdoes not exhibit substantial activity in other gut cells or in a widevariety of other tissues.

In a number of preferred embodiments, an expression control region of atherapeutic construct comprises a gut endocrine cell-specific promoter.Especially preferred are regulatable endocrine cell-specific promoters.The GIP promoter is a specific example of a regulatable gut endocrinecell promoter (see U.S. Ser. No. 09/804,409 which is expresslyincorporated herein in it's entirety by reference). The GIP-promoter isglucose-regulatable and can confer glucose-regulatable endocrinecell-specific expression to an operably linked therapeutic nucleic acid.

Additional examples of gut-specific promoters that may be employed inthe invention are listed in Table 2. Many of these promoters are alsoregulatable. This list is merely exemplary and is not intended to beexhaustive of all the possible gut-specific promoters useful in theinvention.

TABLE 2 Exemplary Promoters and Enhancers for Targeting Expression ofProteins to Endocrine Cells in the Gut Exemplary Promoters and Enhancersfor Targeting Expression of Proteins to Endocrine Cells in the GutGlucokinase Chromogranin A and B Glucose-dependent InsulinotropicPolypeptide Cholycystokinin Proglucagon Adenosine deaminase SecretinGastrin Somatostatin Motilin Ghrelin Sucrose-isomaltase

Preferred expression control regions confer regulatable expression to anoperably linked therapeutic nucleic acid. A signal (sometimes referredto as a stimulus) can increase or decrease expression of a therapeuticnucleic acid operably linked to such an expression control region. Suchexpression control regions that increase expression in response to asignal are often referred to as inducible. Such expression controlregions that decrease expression in response to a signal are oftenreferred to as repressible. Typically, the amount of increase ordecrease conferred by such elements is proportional to the amount ofsignal present; the greater the amount of signal, the greater theincrease or decrease in expression.

Numerous regulatable promoters are known in the art. Preferred inducibleexpression control regions include those comprising an induciblepromoter that is stimulated with a small molecule chemical compound. Inone embodiment, an expression control region is responsive to a chemicalthat is orally deliverable but not normally found in food. Particularexamples can be found, for example, in U.S. Pat. Nos. 5,989,910;5,935,934; 6,015,709; and 6,004,941.

A particularly preferred expression control region is one that increasesor decreases expression of an operably linked therapeutic nucleic acidin response to the presence of a nutrient, in which case the expressioncontrol region is referred to as “nutrient-regulatable.” Anutrient-regulatable expression control region generally provides basallevels of transcription in the absence of the nutrient. Typically, basallevels of transcription are greater for a repressible element than foran inducible element.

The term “nutrient” is used broadly to refer to any of the organic orinorganic substances present in ingestible or consumable material.Particular examples of nutrients include sugars (e.g., glucose, lactose,sucrose, fructose, mannose, etc.), carbohydrates, starches, fats(saturated or unsaturated), lipids, fatty acids, triglycerides,polypeptides, amino acids, cellulose, hormones, vitamins, and minerals.

Nutrient-regulatable expression control regions exist, for example, aspromoters that regulate expression of enzymes involved in glycolysis,lipid metabolism, carbohydrate metabolism and cholesterol (e.g.,steroid) metabolism, which are modulated by sugars, fats, carbohydrate,and cholesterol, respectively, and are applicable in the invention.Particular examples of nutrient-regulatable elements are glucoseinducible elements that drive expression of L-pyruvate kinase,acetyl-CoA-carboxylase, spot-14, fatty acid synthase, glyceraldehydephosphate dehydrogenase phospho-enol-pyruvate carboxykinase,glucose-6-phosphatase and phosphofructokinase (see, also, e.g., Rutter,G A et al., News Physiol Sci. 15:149 (2000)). Another example of anutrient-regulatable element is the alcohol-dehydrogenase generegulatory element. Yet another example of a nutrient-regulatableelement is the vitamin-D response element, which confers expression inthe presence of vitamin D. The mammalian metallothionein gene promoteris an expression control element inducible by metals. As withtissue-specific control elements, nutrient-regulatable control elementsmay be responsive to multiple nutrients. For example, aglucose-inducible element may also be responsive to lactose. Aparticular nutrient (e.g., glucose) is therefore not meant to beexclusive of other nutrients in that other nutrients may modulateactivity (increase or decrease), to a lesser degree, of the controlelement.

Expression control elements included herein can be from bacteria, yeast,plant, or animal (mammalian or non-mammalian). Thus, any expressioncontrol element from any organism that is inducible by a signal (e.g.,nutrient) in the context of a mammalian gut mucosal cell, preferably ahuman gut mucosal cell, can be used.

Expression control regions include full-length promoter sequences, suchas native promoter and enhancer elements, as well as subsequences orpolynucleotide variants which retain all or part of full-length ornon-variant function (e.g., retain some amount of nutrient regulation orcell/tissue-specific expression). As used herein, the term “functional”and grammatical variants thereof, when used in reference to a nucleicacid sequence, subsequence or fragment, means that the sequence has oneor more functions of native nucleic acid sequence (e.g., non-variant orunmodified sequence). As used herein, the term “variant” means asequence substitution, deletion, or addition, or other modification(e.g., chemical derivatives such as modified forms resistant tonucleases).

As used herein, the term “operable linkage” refers to a physicaljuxtaposition of the components so described as to permit them tofunction in their intended manner. In the example of an expressioncontrol element in operable linkage with a nucleic acid, therelationship is such that the control element modulates expression ofthe nucleic acid. Typically, an expression control region that modulatestranscription is juxtaposed near the 5′ end of the transcribed nucleicacid (i.e., “upstream”). Expression control regions can also be locatedat the 3′ end of the transcribed sequence (i.e., “downstream”) or withinthe transcript (e.g., in an intron). Expression control elements can belocated at a distance away from the transcribed sequence (e.g., 100 to500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleicacid). A specific example of an expression control element is apromoter, which is usually located 5′ of the transcribed sequence.Another example of an expression control element is an enhancer, whichcan be located 5′ or 3′ of the transcribed sequence, or within thetranscribed sequence.

In a number of preferred embodiments, the therapeutic nucleic acid ofthe nanoparticle encodes a therapeutic protein. In an especiallypreferred embodiment, the therapeutic protein is a secreted therapeuticprotein. Preferably, the nanoparticle is capable of increasing thesystemic level of the secreted therapeutic protein. Preferably, thenanoparticle is capable of producing a long-term increase in thesystemic level of the secreted therapeutic protein. In one embodiment,the long-term increase in the systemic level of secreted therapeuticprotein is static. In a preferred embodiment, the long-term increase inthe systemic level of secreted therapeutic protein is dynamic.

It will be appreciated that dynamic levels of therapeutic protein may beachieved with constitutive as well as non-constitutive expressioncontrol regions. In a number of preferred embodiments, therapeuticprotein is stored in gut endocrine cells and released in response to asignal, thereby producing a dynamic systemic level of therapeuticprotein irrespective of whether transcriptional activity is constitutiveor non-constitutive.

Integration

In a number of preferred embodiments, a chitosan-based nanoparticlecomprises a therapeutic construct that comprises (i) a therapeuticnucleic acid operably linked to an expression control region, and (ii)an integration sequence. The integration sequence provides forintegration of the therapeutic nucleic acid operably linked to theexpression control region, taken together, into the genome of a gutmucosal cell. Such a therapeutic construct, when combined in a gutmucosal cell with a means for integration, yields a gut mucosal cellwith a modified genome carrying the heterologous expression controlsequence operably linked to a therapeutic nucleic acid. Accordingly,such nanoparticles provide for the production of gut mucosal cellscomprising heterologous expression control sequences operably linked totherapeutic nucleic acids. Long-term expression extending weeks and evenmonths may be achieved with such nanoparticles.

Numerous means for integration, and integration sequences that functiontherewith, are known in the art (see for example Nunes-Duby et al.,Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol.,165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al.,TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol.,43:413-439, 2003). These include recombinases and transposases. Examplesinclude Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981),lambda (Nash, Nature, 247, 543-545, 1974), Flp (Broach, et al, Cell,29:227-234, 1982) R (Matsuzaki, et al, J. Bacteriology, 172:610-618,1990), φC31 (see for example Groth et al., J. Mol. Biol. 335:667-678,2004; Calos, Curr Gene Ther., 6:633-45, 2006), sleeping beauty,transposases of the mariner family (Plasterk et al., supra), and meansfrom integrating viruses such as AAV, retroviruses, and lentiviruseshaving components that provide for virus integration such as the LTRsequences of retroviruses or lentivirus and the ITR sequences of AAV(Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003).

In one embodiment, a therapeutic construct comprises a singleintegration sequence operably linked to the therapeutic nucleicacid-expression control region polynucleotide. In another embodiment, atherapeutic construct comprises a first and a second integrationsequence, which are operably linked to the therapeutic nucleicacid-expression control region polynucleotide. In the methods herein,the means for integration and the type of integration sequence used arecompatible.

In a number of preferred embodiments, a chitosan-based nanoparticlecomprises a non-therapeutic construct, which non-therapeutic constructcomprises a nucleic acid encoding a means for integration. Anintegration sequence operably linked to a therapeutic nucleicacid-expression control region polynucleotide, when combined in a cellwith a means for integration, provides for genomic insertion of thetherapeutic nucleic acid-expression control region polynucleotide.

Therapeutic Nucleic Acids

As used herein, therapeutic nucleic acids are nucleic acids capable ofexerting a therapeutic effect when expressed in a gut mucosal cell. Thetherapeutic effect exerted by a therapeutic nucleic acid need not be ingut tissue.

Therapeutic nucleic acids include nucleic acids encoding therapeuticproteins, as well as nucleic acids that produce transcripts that aretherapeutic RNAs. A therapeutic RNA is an RNA molecule capable ofexerting a therapeutic effect in a mammalian cell. Therapeutic RNAsinclude antisense RNAs, siRNAs, short hairpin RNAs, and enzymatic RNAs.

As used herein, therapeutic nucleic acids that encode therapeuticproteins include nucleic acids that encode proprotein forms oftherapeutic proteins that require processing, e.g., proteolyticprocessing by a convertase, for activity.

In one embodiment, a therapeutic nucleic acid does not encode a foodallergen. By food allergen is meant a food allergen protein that causesa hypersensitivity reaction. Generally, the use of nucleic acidsencoding such food allergen proteins is undesirable because of thepotentially lethal consequences of expression.

In one embodiment, a therapeutic nucleic acid encodes an immunogen thatis capable of eliciting a specific antibody response and/or T cellresponse to the immunogen. Preferably, the immunogen is capable ofeliciting a therapeutic immune response.

The nature of therapeutic proteins and encoding therapeutic nucleicacids that are useful in the present invention is not constrained by thechitosan-based nanoparticles of the invention. A wide variety oftherapeutic nucleic acids of wide ranging sizes are contemplated foruse. Especially preferred for use in the invention are therapeuticnucleic acids encoding secretable therapeutic proteins, which may besecreted into the blood circulatory system by gut endocrine cells.

As used herein, the term “produces” or “production,” when used inreference to a secreted therapeutic protein refers to expression orsecretion of the protein by a gut mucosal cell. In some embodiments, asignal or stimuli (i.e., a secretagogue) stimulates release into thesystemic circulation of a secretable therapeutic protein already presentin the cell.

In some preferred embodiments, a therapeutic nucleic acid encoding asecreted therapeutic protein is expressed in a gut endocrine cell (e.g.,K-cell, L-cell, etc.). The expression control region used to conferexpression of the therapeutic protein may or may not be regulatable, butin either case a signal typically will regulate secretion of the proteinfrom the cell into the blood. In this case, the signal or stimulifunctions as a secretagogue that stimulates or increases secretion of aprotein.

In one embodiment, therapeutic nucleic acids are engineered to encodesecretable proforms of therapeutic proteins that are not processedwithin gut cells. Rather, these proforms (protherapeutics) are activatedin the vicinity of target tissues.

Methods and therapeutic nucleic acids for the treatment of diseasesassociated with gut mucosa are also contemplated by the presentinvention. For example, compositions and methods for the treatment ofcancer and/or inflammation associated with gut mucosa are contemplated.

Therapeutic proteins contemplated for use in the invention have a widevariety of activities and find use in the treatment of a wide variety ofdisorders. The following description of therapeutic protein activities,and indications treatable with therapeutic proteins of the invention, isexemplary and not intended to be exhaustive. The term “subject” refersto an animal, with mammals being preferred, and humans being especiallypreferred.

A partial list of therapeutic proteins and target diseases is shown inTable 3.

TABLE 3 THERAPEUTIC LEAD COMPOUNDS TARGET DISEASE FUNCTION EFFECTInsulin Diabetes Insulin replacement Improve glucose tolerance.Delay/prevent diabetes. Glucagon antagonists Diabetes Reduce endogenousImprove glucose glucose production tolerance GLP-1 Diabetes Stimulategrowth of β- Improve glucose Obesity cells, improve insulin tolerance.sensitivity, suppress Induce weight loss appetite Leptin ObesityAppetite suppression Induce weight loss. Diabetes and improvement ofImprove glucose insulin sensitivity tolerance CCK Obesity Appetitesuppression Induce weight loss Growth Hormone (GH) GH deficiencies, GHreplacement Improve growth wasting and anti-aging Clotting factorsHemophilia Clotting factors Improve clotting time replacementTherapeutic antibodies Infections Pathogen Prevent infections or andantibody Cancer neutralization or transplant rejectionsfragments/portions immune modulations inflammation inhibitors, GI tractinflammation; immune modulation prevent inflammation in e.g., IL-10,TNFα e.g., inflammatory GI tract antagonists, IL-17 bowel diseaseantagonists

Hyperglycemia and Body Mass

Therapeutic proteins include insulin and insulin analogs. Diabetesmellitus is a debilitating metabolic disease caused by absent (type 1)or insufficient (type 2) insulin production from pancreatic β-cells(Unger, R. H. et al., Williams Textbook of Endocrinology Saunders,Philadelphia (1998)). Beta-cells are specialized endocrine cells thatmanufacture and store insulin for release following a meal (Rhodes, et.al. J. Cell Biol. 105:145 (1987)) and insulin is a hormone thatfacilitates the transfer of glucose from the blood into tissues where itis needed. Patients with diabetes must frequently monitor blood glucoselevels and many require multiple daily insulin injections to survive.However, such patients rarely attain ideal glucose levels by insulininjection (Turner, R. C. et al. JAMA 281:2005 (1999)). Furthermore,prolonged elevation of insulin levels can result in detrimental sideeffects such as hypoglycemic shock and desensitization of the body'sresponse to insulin. Consequently, diabetic patients still developlong-term complications, such as cardiovascular diseases, kidneydisease, blindness, nerve damage and wound healing disorders (UKProspective Diabetes Study (UKPDS) Group, Lancet 352, 837 (1998)).

Disorders treatable by a method of the invention include a hyperglycemiccondition, such as insulin-dependent (type 1) or -independent (type 2)diabetes, as well as physiological conditions or disorders associatedwith or that result from the hyperglycemic condition. Thus,hyperglycemic conditions treatable by a method of the invention alsoinclude a histopathological change associated with chronic or acutehyperglycemia (e.g., diabetes). Particular examples include degenerationof pancreas (β-cell destruction), kidney tubule calcification,degeneration of liver, eye damage (diabetic retinopathy), diabetic foot,ulcerations in mucosa such as mouth and gums, excess bleeding, delayedblood coagulation or wound healing and increased risk of coronary heartdisease, stroke, peripheral vascular disease, dyslipidemia, hypertensionand obesity.

Thus, in various methods of the invention, a gut mucosal cell thatproduces insulin or a functional subsequence of insulin or an analog ofinsulin in response to glucose is useful for decreasing glucose,improving glucose tolerance, treating a hyperglycemic condition (e.g.,diabetes) or for treating a physiological disorders associated with orresulting from a hyperglycemic condition. Such disorders include, forexample, diabetic neuropathy (autonomic), nephropathy (kidney damage),skin infections and other cutaneous disorders, slow or delayed healingof injuries or wounds (e.g., that lead to diabetic carbuncles), eyedamage (retinopathy, cataracts) which can lead to blindness, diabeticfoot and accelerated periodontitis. Such disorders also includeincreased risk of developing coronary heart disease, stroke, peripheralvascular disease, dyslipidemia, hypertension and obesity.

As used herein, the term “hyperglycemic” or “hyperglycemia,” when usedin reference to a condition of a subject, means a transient or chronicabnormally high level of glucose present in the blood of a subject. Thecondition can be caused by a delay in glucose metabolization orabsorption such that the subject exhibits glucose intolerance or a stateof elevated glucose not typically found in normal subjects (e.g., inglucose-intolerant subdiabetic subjects at risk of developing diabetes,or in diabetic subjects). Fasting plasma glucose (FPG) levels fornormoglycemia are less than about 110 mg/dl, for impaired glucosemetabolism, between about 110 and 126 mg/dl, and for diabetics greaterthan about 126 mg/dl.

Disorders treatable by producing a protein in a gut mucosal tissue alsoinclude obesity or an undesirable body mass. Leptin, cholecystokinin,PYY and GLP-1 decrease hunger, increase energy expenditure, induceweight loss or provide normal glucose homeostasis. Thus, in variousembodiments, a method of the invention for treating obesity or anundesirable body mass, or hyperglycemia, involves the use of atherapeutic nucleic acid encoding leptin, cholecystokinin, PYY or GLP-1.Disorders treatable also include those typically associated withobesity, for example, abnormally elevated serum/plasma LDL, VLDL,triglycerides, cholesterol, plaque formation leading to narrowing orblockage of blood vessels, increased risk of hypertension/stroke,coronary heart disease, etc.

As used herein, the term “obese” or “obesity” refers to a subject havingat least a 30% increase in body mass in comparison to an age and gendermatched normal subject. “Undesirable body mass” refers to subjectshaving 1%-29% greater body mass than a matched normal subject as well assubjects that are normal with respect to body mass but who wish todecrease or prevent an increase in their body mass.

In one embodiment, a therapeutic protein of the invention is a glucagonantagonist. Glucagon is a peptide hormone produced by α-cells inpancreatic islets and is a major regulator of glucose metabolism (UngerR. H. & Orci L. N. Eng. J. Med. 304:1518 (1981); Unger R. H. Diabetes25:136 (1976)). As with insulin, blood glucose concentration mediatesglucagon secretion. However, in contrast to insulin glucagon is secretedin response to a decrease in blood glucose. Therefore, circulatingconcentrations of glucagon are highest during periods of fast and lowestduring a meal. Glucagon levels increase to curtail insulin frompromoting glucose storage and stimulate liver to release glucose intothe blood. A specific example of a glucagon antagonist is [des-His¹,des-Phe⁶, Glu⁹]glucagon-NH₂. In streptozotocin diabetic rats, bloodglucose levels were lowered by 37% within 15 min of an intravenous bolus(0.75 μg/g body weight) of this glucagon antagonist (Van Tine B. A. et.al. Endocrinology 137:3316 (1996)).

In another embodiment, a therapeutic protein of the invention useful fortreating a hyperglycemic condition or undesirable body mass (e.g.,obesity) is a glucagon-like peptide-1 (GLP-1). GLP-1 is a hormonereleased from L-cells in the intestine during a meal which stimulatespancreatic β-cells to increase insulin secretion. GLP-1 has additionalactivities which make it an attractive therapeutic agent for treatingobesity and diabetes. For example, GLP-1 reduces gastric emptying,suppresses appetite, reduces glucagon concentration, increases.beta.-cell mass, stimulates insulin biosynthesis and secretion in aglucose-dependent fashion, and likely increases tissue sensitivity toinsulin (Kieffer T. J., Habener J. F. Endocrin. Rev. 20:876 (2000)).Therefore, regulated release of GLP-1 in the gut to coincide with a mealcan provide therapeutic benefit for a hyperglycemic condition or anundesirable body mass. GLP-1 analogs that are resistant to dipeptidylpeptidate IV (DPP IV) provide longer duration of action and improvedtherapeutic value. Thus, GLP-1 analogs are preferred therapeuticpolypeptides.

In another embodiment, a therapeutic protein of the invention useful fortreating a hyperglycemic condition is an antagonist to the hormoneresistin. Resistin is an adipocyte-derived factor for which expressionis elevated in diet-induced and genetic forms of obesity. Neutralizationof circulating resistin improves blood glucose and insulin action inobese mice. Conversely, administration of resistin in normal miceimpairs glucose tolerance and insulin action (Steppan C M et. al. Nature409:307 (2001)). Production of a protein that antagonizes the biologicaleffects of resistin in gut can therefore provide an effective therapyfor obesity-linked insulin resistance and hyperglycemic conditions.

In another embodiment, a therapeutic polypeptide of the invention usefulfor treating a hyperglycemic condition or undesirable body mass (e.g.,obesity) is leptin. Leptin, although produced primarily by fat cells, isalso produced in smaller amounts in a meal-dependent fashion in thestomach. Leptin relays information about fat cell metabolism and bodyweight to the appetite centers in the brain where it signals reducedfood intake (promotes satiety) and increases the body's energyexpenditure.

In another embodiment, a therapeutic polypeptide of the invention usefulfor treating a hyperglycemic condition or undesirable body mass (e.g.,obesity) is the C-terminal globular head domain of adipocytecomplement-related protein (Acrp30). Acrp30 is a protein produced bydifferentiated adipocytes. Administration of a proteolytic cleavageproduct of Acrp30 consisting of the globular head domain to mice leadsto significant weight loss (Fruebis J. et al. Proc. Natl. Acad. Sci. USA98:2005 (2001)).

In another embodiment, a therapeutic polypeptide of the invention usefulfor treating a hyperglycemic condition or undesirable body mass (e.g.,obesity) is cholecystokinin (CCK). CCK is a gastrointestinal peptidesecreted from the intestine in response to particular nutrients in thegut. CCK release is proportional to the quantity of food consumed and isbelieved to signal the brain to terminate a meal (Schwartz M. W. et. al.Nature 404:661-71 (2000)). Consequently, elevated CCK can reduce mealsize and promote weight loss or weight stabilization (i.e., prevent orinhibit increases in weight gain).

Regarding PYY, see for example 1e Roux et al., Proc Nutr Soc. 2005 May;64(2):213-6.

Immunological Disorders

In one embodiment, a therapeutic protein of the invention possessesimmunomodulatory activity. For example, a therapeutic polypeptide of thepresent invention may be useful in treating deficiencies or disorders ofthe immune system, by activating or inhibiting the proliferation,differentiation, or mobilization (chemotaxis) of immune cells. Immunecells develop through the process of hematopoiesis, producing myeloid(platelets, red blood cells, neutrophils, and macrophages) and lymphoid(B and T lymphocytes) cells from pluripotent stem cells. The etiology ofthese immune deficiencies or disorders may be genetic, somatic, such ascancer or some autoimmune disorders, acquired (e.g. by chemotherapy ortoxins), or infectious.

A therapeutic polypeptide of the present invention may be useful intreating deficiencies or disorders of hematopoietic cells. A therapeuticpolypeptide of the present invention could be used to increasedifferentiation or proliferation of hematopoietic cells, including thepluripotent stem cells, in an effort to treat those disorders associatedwith a decrease in certain (or many) types hematopoietic cells. Examplesof immunologic deficiency syndromes include, but are not limited to:blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

A therapeutic polypeptide of the present invention may also be useful intreating autoimmune disorders. Many autoimmune disorders result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration of atherapeutic polypeptide of the present invention that inhibits an immuneresponse, particularly the proliferation, differentiation, or chemotaxisof T-cells, may be an effective therapy in preventing autoimmunedisorders.

Examples of autoimmune disorders that can be treated by the presentinvention include, but are not limited to: Addison's Disease, hemolyticanemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis,allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome,Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis,Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies,Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis,Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,Guillain-Barre Syndrome, insulin-dependent diabetes mellitis, Crohn'sdisease, ulcerative colitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by a therapeutic polypeptide of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

A therapeutic polypeptide of the present invention may also be used totreat and/or prevent organ rejection or graft-versus-host disease(GVHD). Organ rejection occurs by host immune cell destruction of thetransplanted tissue through an immune response. Similarly, an immuneresponse is also involved in GVHD, but, in this case, the foreigntransplanted immune cells destroy the host tissues. The administrationof a therapeutic polypeptide of the present invention that inhibits animmune response, particularly the proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD.

Similarly, a therapeutic polypeptide of the present invention may alsobe used to modulate inflammation. For example, the therapeuticpolypeptide may inhibit the proliferation and differentiation of cellsinvolved in an inflammatory response. These molecules can be used totreat inflammatory conditions, both chronic and acute conditions,including inflammation associated with infection (e.g. septic shock,sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g. TNF orIL-1.)

Clotting Disorders

In some embodiments, a therapeutic polypeptide of the present inventionmay also be used to modulate hemostatic (the stopping of bleeding) orthrombolytic activity (clot formation). For example, by increasinghemostatic or thrombolytic activity, a therapeutic polypeptide of thepresent invention could be used to treat blood coagulation disorders(e.g. afibrinogenemia, factor deficiencies), blood platelet disorders(e.g. thrombocytopenia), or wounds resulting from trauma, surgery, orother causes. Alternatively, a therapeutic polypeptide of the presentinvention that can decrease hemostatic or thrombolytic activity could beused to inhibit or dissolve clotting. These molecules could be importantin the treatment of heart attacks (infarction), strokes, or scarring. Inone embodiment, a therapeutic polypeptide of the invention is a clottingfactor, useful for the treatment of hemophilia or othercoagulation/clotting disorders (e.g., Factor VIII, IX or X)

Hyperproliferative Disorders

In one embodiment, a therapeutic protein of the invention is capable ofmodulating cell proliferation. Such a therapeutic polypeptide can beused to treat hyperproliferative disorders, including neoplasms.

Examples of hyperproliferative disorders that can be treated by atherapeutic polypeptide of the present invention include, but are notlimited to neoplasms located in the: abdomen, bone, breast, digestivesystem, liver, pancreas, peritoneum, endocrine glands (adrenal,parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, headand neck, nervous (central and peripheral), lymphatic system, pelvic,skin, soft tissue, spleen, thoracic, and urogenital.

Similarly, other hyperproliferative disorders can also be treated by atherapeutic polypeptide of the present invention. Examples of suchhyperproliferative disorders include, but are not limited to:hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,Gaucher's Disease, histiocytosis, and any other hyperproliferativedisease, besides neoplasia, located in an organ system listed above.

A therapeutic polypeptide produced in gut mucosa according to thepresent invention may inhibit the proliferation of the disorder throughdirect or indirect interactions. Delivery to the circulatory systemprovides for access of therapeutic protein to a wide variety of tissues.Alternatively, a therapeutic polypeptide of the present invention maystimulate the proliferation of other cells which can inhibit thehyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as with a chemotherapeutic agent.

Infectious Disease

In one embodiment, a therapeutic polypeptide of the present inventioncan be used to treat infectious disease. For example, by increasing theimmune response, particularly increasing the proliferation anddifferentiation of B and/or T cells, infectious diseases may be treated.The immune response may be increased by either enhancing an existingimmune response, or by initiating a new immune response. Alternatively,the therapeutic polypeptide of the present invention may also directlyinhibit the infectious agent, without necessarily eliciting an immuneresponse.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated by a therapeutic polypeptide of the presentinvention. Examples of viruses, include, but are not limited to thefollowing DNA and RNA viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis),Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),Mononegavirus (e.g. Paramyxoviridae, Morbillivirus, Rhabdoviridae),Orthomyxoviridae (e.g. Influenza), Papovaviridae, Parvoviridae,Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae(e.g. Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), andTogaviridae (e.g. Rubivirus). Viruses falling within these families cancause a variety of diseases or symptoms, including, but not limited to:arthritis, bronchiollitis, encephalitis, eye infections (e.g.conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B,C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g.AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever,Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia,Rubella, sexually transmitted diseases, skin diseases (e.g. Kaposi's,warts), and viremia. A therapeutic polypeptide of the present inventioncan be used to treat any of these symptoms or diseases.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated or detected by a therapeutic polypeptide of thepresent invention include, but are not limited to, the followingGram-Negative and Gram-positive bacterial families and fungi:Actinomycetales (e.g. Corynebacterium, Mycobacterium, Norcardia),Aspergillosis, Bacillaceae (e.g. Anthrax, Clostridium), Bacteroidaceae,Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses,Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia),Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria,Mycoplasmatales, Neisseriaceae (e.g. Acinetobacter, Gonorrhea,Menigococcal), Pasteurellacea Infections (e.g. Actinobacillus,Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae,Syphilis, and Staphylococcal. These bacterial or fungal families cancause the following diseases or symptoms, including, but not limited to:bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis,uveitis), gingivitis, opportunistic infections (e.g. AIDS relatedinfections), paronychia, prosthesis-related infections, Reiter'sDisease, respiratory tract infections, such as Whooping Cough orEmpyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery,Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea,meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g.cellulitis, dermatocycoses), toxemia, urinary tract infections, woundinfections. A therapeutic polypeptide of the present invention can beused to treat any of these symptoms or diseases.

Moreover, parasitic agents causing disease or symptoms that can betreated by a therapeutic polypeptide of the present invention include,but are not limited to, the following families: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas. These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease (e.g. dysentery,giardiasis), liver disease, lung disease, opportunistic infections (e.g.AIDS related), Malaria, pregnancy complications, and toxoplasmosis. Atherapeutic polypeptide of the present invention can be used to treatany of these symptoms or diseases.

Regeneration

A therapeutic polypeptide of the present invention can be used todifferentiate, proliferate, and attract cells, fostering to theregeneration of tissues. (See, Science 276:59-87 (1997).) Theregeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, burns, incisions,or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated with the contribution of a therapeuticprotein of the invention include organs (e.g. pancreas, liver,intestine, kidney, skin, endothelium), muscle (smooth, skeletal orcardiac), vascular (including vascular endothelium), nervous,hematopoietic, and skeletal (bone, cartilage, tendon, and ligament)tissue. Preferably, regeneration incurs a small amount of scarring, oroccurs without scarring. Regeneration also may include angiogenesis.

Moreover, a therapeutic polypeptide of the present invention mayincrease regeneration of tissues difficult to heal. For example,increased tendon/ligament regeneration would quicken recovery time afterdamage. A therapeutic polypeptide of the present invention could also beused prophylactically in an effort to avoid damage. Specific diseasesthat could be treated include tendinitis, carpal tunnel syndrome, andother tendon or ligament defects. A further example of tissueregeneration of non-healing wounds includes pressure ulcers, ulcersassociated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using atherapeutic polypeptide of the present invention to proliferate anddifferentiate nerve cells. Diseases that could be treated using thismethod include central and peripheral nervous system diseases,neuropathies, or mechanical and traumatic disorders (e.g. spinal corddisorders, head trauma, cerebrovascular disease, and stoke).Specifically, diseases associated with peripheral nerve injuries,peripheral neuropathy (e.g. resulting from chemotherapy or other medicaltherapies), localized neuropathies, and central nervous system diseases(e.g. Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all betreated using therapeutic proteins of the present invention. Withrespect to CNS disorders, numerous means are known in the art forfacilitating therapeutic access to brain tissue, including methods fordisrupting the blood brain barrier, and methods of coupling therapeuticagents to moieties that provide for transport into the CNS. In oneembodiment, a therapeutic nucleic acid is engineered so as to encode afusion protein, which fusion protein comprises a transport moiety and atherapeutic protein.

Chemotaxis

In one embodiment, a therapeutic polypeptide of the present inventionpossesses a chemotaxis activity. A chemotaxic molecule attracts ormobilizes cells (e.g. monocytes, fibroblasts, neutrophils, T-cells, mastcells, eosinophils, epithelial and/or endothelial cells) to a particularsite in the body, such as inflammation, infection, or site ofhyperproliferation. The mobilized cells can then fight off and/or healthe particular trauma or abnormality.

A therapeutic polypeptide of the present invention may increasechemotaxic activity of particular cells. These chemotactic molecules canthen be used to treat inflammation, infection, hyperproliferativedisorders, or any immune system disorder by increasing the number ofcells targeted to a particular location in the body. For example,chemotaxic molecules can be used to treat wounds and other trauma totissues by attracting immune cells to the injured location. Chemotacticmolecules of the present invention can also attract fibroblasts, whichcan be used to treat wounds.

It is also contemplated that a therapeutic polypeptide of the presentinvention may inhibit chemotactic activity. These molecules could alsobe used to treat disorders. Thus, a therapeutic polypeptide of thepresent invention could be used as an inhibitor of chemotaxis.

Especially preferred for use are protherapeutic proteins that areactivated in the vicinity of target tissues.

Additional therapeutic polypeptides contemplated for use include, butare not limited to, growth factors (e.g., growth hormone, insulin-likegrowth factor-1, platelet-derived growth factor, epidermal growthfactor, acidic and basic fibroblast growth factors, transforming growthfactor-β, etc.), to treat growth disorders or wasting syndromes; andantibodies (e.g., human or humanized), to provide passive immunizationor protection of a subject against foreign antigens or pathogens (e.g.,H. Pylori), or to provide treatment of cancer, arthritis orcardiovascular disease; cytokines, interferons (e.g., interferon (INF),INF-α2b and 2a, INF-αN1, INF-β1b, INF-gamma), interleukins (e.g., IL-1to IL-10), tumor necrosis factor (TNF-α TNF-β), chemokines, granulocytemacrophage colony stimulating factor (GM-CSF), polypeptide hormones,antimicrobial polypeptides (e.g., antibacterial, antifungal, antiviral,and/or antiparasitic polypeptides), enzymes (e.g., adenosine deaminase),gonadotrophins, chemotactins, lipid-binding proteins, filgastim(Neupogen), hemoglobin, erythropoietin, insulinotropin, imiglucerase,sarbramostim, tissue plasminogen activator (tPA), urokinase,streptokinase, phenylalanine ammonia lyase, brain-derived neurotrophicfactor (BDNF), nerve growth factor (NGF), thrombopoietin (TPO),superoxide dismutase (SOD), adenosine deamidase, catalase calcitonin,endothelian, L-asparaginase pepsin, uricase trypsin, chymotrypsinelastase, carboxypeptidase lactase, sucrase intrinsic factor, calcitoninparathyroid hormone(PTH)-like, hormone, soluble CD4, and antibodiesand/or antigen-binding fragments (e.g, FAbs) thereof (e.g., orthocloneOKT-e (anti-CD3), GPIIb/IIa monoclonal antibody).

Methods for Transfecting Gut Mucosal Cells

In one aspect, the invention provides methods for transfecting gutmucosal cells with therapeutic nucleic acids in vivo. The methodscomprise contacting gut mucosa in vivo with a chitosan-basednanoparticle of the invention.

The methods are used to transfect gut mucosal cells in mammals. In apreferred embodiment, the mammal is a primate. In an especiallypreferred embodiment, the mammal is a human. In another embodiment, themammal is a non-primate. Preferred non-primate mammals include dogs,cats and horses.

In a preferred embodiment, the method involves contacting mucosa of thesmall intestine. In a preferred embodiment, the method involvescontacting mucosa of the duodenum, jejunum, or ileum with achitosan-based nanoparticle of the invention.

In a preferred embodiment, the method involves contacting mucosa of thestomach with a chitosan-based nanoparticle of the invention.

In a preferred embodiment, the method involves contacting mucosa of thecolon with a chitosan-based nanoparticle of the invention.

In a preferred embodiment, the chitosan-based nanoparticle transfects agut mucosal precursor cell. In a preferred embodiment, the gut mucosalprecursor cell is a gut endocrine cell precursor cell. In a preferredembodiment, the gut endocrine cell precursor cell produces a gutendocrine cell selected from the group consisting of K cells, L-cells,S-cells, G-cells, D-cells, I-cells, Mo-cells, and Gr-cells. In anespecially preferred embodiment, the gut endocrine cell precursor cellproduces a K cell.

In a preferred embodiment, the gut mucosal precursor cell is a mucosalcell of the small intestine. In a preferred embodiment, the gut mucosalprecursor cell is a mucosal cell of the duodenum, jejunum, or ileum.

In a preferred embodiment, the gut mucosal precursor cell is a mucosalcell of the stomach.

In a preferred embodiment, the gut mucosal precursor cell is a mucosalcell of the colon.

In a preferred embodiment, the gut mucosal precursor cell produces amucosal cell that expresses the therapeutic nucleic acid.

In one embodiment, the therapeutic nucleic acid encodes a therapeuticRNA.

In a preferred embodiment, the therapeutic nucleic acid encodes atherapeutic protein. In a preferred embodiment, the therapeutic proteinis a secreted therapeutic protein.

In a preferred embodiment, the therapeutic nucleic acid of thenanoparticle encodes a therapeutic protein that is selected from thegroup consisting of hormones, enzymes, cytokines, chemokines,antibodies, mitogenic factors, growth factors, differentiation factors,factors influencing angiogenesis, factors influencing blood clotformation, factors influencing blood glucose levels, glucose metabolism,factors influencing lipid metabolism, factors influencing bloodcholesterol levels, factors influencing blood LDL or HDL levels, factorsinfluencing cell apoptosis, factors influencing food intake, factorsinfluencing energy expenditure, factors influencing appetite, factorsinfluencing nutrient absorption, factors influencing inflammation andfactors influencing bone formation. Particularly preferred aretherapeutic nucleic acids encoding insulin, leptin, glucagon antagonist,GLP-1, GLP-2, Ghrelin, cholecystokinin, growth hormone, clottingfactors, PYY, erythropoietin, inhibitors of inflammation, IL-10, IL-17antagonists, TNFα antagonists, growth hormone releasing hormone,parathyroid hormone. In a preferred embodiment, the encoded therapeuticprotein is insulin. In another preferred embodiment, the encodedtherapeutic protein is an insulin analog. In another preferredembodiment, the encoded therapeutic protein is leptin. In anotherpreferred embodiment, the encoded therapeutic protein is PYY.

In a preferred embodiment, the therapeutic protein is produced in a gutmucosal cell and enters the systemic circulation such that the systemiclevel of the therapeutic protein is increased. In a preferredembodiment, the therapeutic protein is released by regulated secretioninto the systemic circulation.

In one embodiment, the systemic level of the therapeutic protein isincreased by at least about 10 pM, more preferably by at least about 100pM, more preferably by at least 1 nM, more preferably by at least about10 nM.

In one embodiment, the systemic level of the therapeutic protein isincreased by at least 10-fold, more preferably at least 100-fold, morepreferably at least 125-fold, more preferably at least 150-fold, morepreferably at least 200-fold, more preferably at least 200-fold, morepreferably at least 500-fold, more preferably at least 750-fold, morepreferably at least 1000-fold higher than the lowest detectableconcentration of the therapeutic protein.

In one embodiment, the systemic level of the therapeutic protein isincreased to at least 10%, more preferably 25%, more preferably 50%,more preferably, 75%, more preferably 100%, more preferably 125%, morepreferably 150%, more preferably 200%, more preferably 500%, morepreferably 750%, and most preferably at least 1000% of a physiologicallyactive concentration.

In a preferred embodiment, the systemic level of the therapeutic proteinis increased for longer than about 4 days, more preferably longer thanabout 5 days, more preferably longer than about 6 days, more preferablylonger than about 7 days, more preferably longer than about 10 days,more preferably longer than about 2 weeks, more preferably longer thanabout 3 weeks, more preferably longer than about 4 weeks, morepreferably longer than about 6 weeks, more preferably longer than about8 weeks, more preferably longer than about 10 weeks, and most preferablylonger than about 12 weeks.

In one embodiment, the increase in the systemic level of the therapeuticprotein is static. In a preferred embodiment, the increase in thesystemic level of the therapeutic protein is dynamic.

In one embodiment, the method comprises contacting gut mucosa in vivowith a first chitosan-based nanoparticle and a second chitosan-basednanoparticle. The first chitosan based nanoparticle is capable oftransfecting a gut mucosal precursor cell in vivo and comprises (i) aplurality of chitosan polymers, and (ii) a therapeutic construct,wherein the therapeutic construct comprises a therapeutic nucleic acidoperably linked to an expression control region functional in a gutmucosal cell, and an integration sequence. The second chitosan-basednanoparticle is capable of transfecting a gut mucosal precursor cell invivo and comprises (i) a plurality of chitosan polymers, and (ii) anon-therapeutic construct, wherein the non-therapeutic constructcomprises a nucleic acid encoding a means for integration operablylinked to an expression control region that is functional in a gutmucosal precursor cell. A gut mucosal precursor cell in the gut mucosais transfected with the first and second nanoparticles. The nucleic acidof the second nanoparticle is expressed in the gut mucosal precursorcell to produce a means for integration, whereby the means forintegration integrates the therapeutic nucleic acid operably linked toan expression control region provided by the first nanoparticle into thegenome of the gut mucosal precursor cell.

In another embodiment, the method comprises contacting gut mucosa invivo with a chitosan-based nanoparticle comprising (i) a plurality ofchitosan polymers; (ii) a therapeutic construct, wherein the therapeuticconstruct comprises a therapeutic nucleic acid operably linked to anexpression control region functional in a gut mucosal cell, and anintegration sequence; and (iii) a non-therapeutic construct, wherein thenon-therapeutic construct comprises a nucleic acid encoding a means forintegration operably linked to an expression control region that isfunctional in a gut mucosal precursor cell. A gut mucosal precursor cellin the gut mucosa is transfected using the nanoparticle. The nucleicacid encoding a means for integration is expressed in the gut mucosalprecursor cell to produce a means for integration, whereby the means forintegration integrates the therapeutic nucleic acid operably linked toan expression control region into the genome of the gut mucosalprecursor cell.

In one aspect, the invention provides methods for increasing thesystemic level of secreted therapeutic proteins in a mammal. The methodscomprise contacting gut mucosa of a mammal with a chitosan-basednanoparticle of the invention, wherein the nanoparticle comprises atherapeutic nucleic acid encoding a secreted therapeutic protein,wherein the secreted therapeutic protein is produced in a mucosal cellof the gut mucosa, and wherein the secreted therapeutic protein producedin the mucosal cell enters the systemic circulation such that thesystemic level of the secreted therapeutic protein is increased.

In one aspect, the invention provides methods for treating patientshaving diseases or conditions treatable by increasing the systemic levelof therapeutic proteins. The methods comprise contacting gut mucosa of apatient with a chitosan-based nanoparticle of the invention, wherein thenanoparticle comprises a therapeutic nucleic acid encoding a secretedtherapeutic protein, wherein the secreted therapeutic protein isproduced in a mucosal cell of the gut mucosa of the patient, and whereinthe secreted therapeutic protein produced in the mucosal cell enters thesystemic circulation such that the systemic level of the secretedtherapeutic protein is increased.

In a preferred embodiment, the disease is a metabolic disease.

In a preferred embodiment, the disease is diabetes mellitus.

In another preferred embodiment, the condition is morbid obesity.

In another preferred embodiment, the condition is growth deficiency.

In a preferred embodiment, the chitosan-based nanoparticle is orallyadministered.

In a preferred embodiment, the chitosan-based nanoparticle isadministered endoscopically.

In a preferred embodiment, the chitosan-based nanoparticle isadministered rectally.

In a preferred embodiment, the mucosal cell is a gut endocrine cell. Ina preferred embodiment, the gut endocrine cell is selected from thegroup consisting of K cells, L-cells, S-cells, G-cells, D-cells,I-cells, Mo-cells, and Gr-cells. In an especially preferred embodiment,the gut endocrine cell is a K cell.

In a preferred embodiment, the mucosal cell is a mucosal cell of thesmall intestine. In a preferred embodiment, the mucosal cell is amucosal cell of the duodenum, jejunum, or ileum.

In a preferred embodiment, the mucosal cell is a mucosal cell of thestomach.

In a preferred embodiment, the mucosal cell is a mucosal cell of thecolon.

In a preferred embodiment, the therapeutic nucleic acid of thenanoparticle encodes a therapeutic protein that is selected from thegroup consisting of hormones, enzymes, cytokines, chemokines,antibodies, mitogenic factors, growth factors, differentiation factors,factors influencing angiogenesis, factors influencing blood clotformation, factors influencing blood glucose levels, factors influencingglucose metabolism, factors influencing lipid metabolism, factorsinfluencing blood cholesterol levels, factors influencing blood LDL orHDL levels, factors influencing cell apoptosis, factors influencing foodintake, factors influencing energy expenditure, factors influencingappetite, factors influencing nutrient absorption, factors influencinginflammation, and factors influencing bone formation. Particularlypreferred are therapeutic nucleic acids encoding insulin, leptin,glucagon antagonist, GLP-1, GLP-2, Ghrelin, cholecystokinin, growthhormone, clotting factors, PYY, erythropoietin, inhibitors ofinflammation, IL-10, IL-17 antagonists, TNFα antagonists, growth hormonereleasing hormone, parathyroid hormone. In a preferred embodiment, theencoded therapeutic protein is insulin. In another preferred embodiment,the encoded therapeutic protein is an insulin analog. In anotherpreferred embodiment, the encoded therapeutic protein is leptin. Inanother preferred embodiment, the encoded therapeutic protein is PYY.

In a preferred embodiment, the secreted therapeutic protein is releasedby regulated secretion from a gut endocrine cell.

In one embodiment, the systemic level of the therapeutic protein isincreased by at least about 10 pM, more preferably by at least about 100pM, more preferably by at least 1 nM, more preferably by at least about10 nM.

In one embodiment, the systemic level of the therapeutic protein isincreased by at least 10-fold, more preferably at least 100-fold, morepreferably at least 125-fold, more preferably at least 150-fold, morepreferably at least 200-fold, more preferably at least 200-fold, morepreferably at least 500-fold, more preferably at least 750-fold, morepreferably at least 1000-fold higher than the lowest detectableconcentration of the therapeutic protein.

In one embodiment, the systemic level of the therapeutic protein isincreased to at least 10%, more preferably 25%, more preferably 50%,more preferably, 75%, more preferably 100%, more preferably 125%, morepreferably 150%, more preferably 200%, more preferably 500%, morepreferably 750%, and most preferably at least 1000% of a physiologicallyactive concentration.

In a preferred embodiment, the systemic level of the secretedtherapeutic protein is increased for longer than about 4 days, morepreferably longer than about 5 days, more preferably longer than about 6days, more preferably longer than about 7 days, more preferably longerthan about 10 days, more preferably longer than about 2 weeks, morepreferably longer than about 3 weeks, more preferably longer than about4 weeks, more preferably longer than about 6 weeks, more preferablylonger than about 8 weeks, more preferably longer than about 10 weeks,and most preferably longer than about 12 weeks.

Treatment generally results in reducing or preventing the severity orsymptoms of the condition in the subject, i.e., an improvement in thesubject's condition or a “therapeutic effect.” Therefore, treatment canreduce the severity or prevent one or more symptoms of the condition oran associated disorder, inhibit progression or worsening of thecondition or an associated disorder, and in some instances, reverse thecondition or an associated disorder. Thus, in the case of ahyperglycemic condition, for example, treatment can reduce bloodglucose, improve glucose tolerance, provide normal glucose homeostasis,or prevent, improve, or reverse a histopathological change associatedwith or that results from the hyperglycemic condition.

Improvement of a histopathological change associated with ahyperglycemic condition includes, for example, preventing further orreducing kidney tubule calcification, decreasing or arrestingretinopathy or cataracts, decreasing wound or injury healing time,reducing diabetic foot, preventing or reducing acceleratedperiodontitis, or decreasing the risk of developing coronary heartdisease, stroke, peripheral vascular disease, dyslipidemia, hypertensionand obesity. Improvement in obesity can include, for example, areduction of body mass or an improvement in an associated disorder, suchas a decrease in cholesterol, LDL or VLDL levels, a decrease in bloodpressure, a decrease in intimal thickening of the blood vesselassociated with high fat diet, a decrease in resting heart rate, anincrease in lung capacity, etc. Improvement in a bleeding disorder, suchas hemophilia can induce, for example, decreased clotting time orfrequency/duration of bleeding episodes.

As used herein, the term “ameliorate” means an improvement in thesubject's condition, a reduction in the severity of the condition, or aninhibition of progression or worsening of the condition. In the case ofa hyperglycemic condition (e.g., diabetes), for example, an improvementcan be a decrease in blood glucose, an increase in insulin, animprovement in glucose tolerance, or glucose homeostasis. An improvementin a hyperglycemic condition also can include improved pancreaticfunction (e.g., inhibit or prevent β-islet cell destruction), a decreasein a pathology associated with or resulting from the condition, such asan improvement in histopathology of an affected tissue or organ, as setforth herein. In the case of obesity, for example, an improvement can bea decrease in weight gain, a reduction of body mass or an improvement ina conditions associated with obesity, as set forth herein (e.g.,reduction of blood glucose, cholesterol, LDL or VLDL levels, a decreasein blood pressure, a decrease in intimal thickening of the blood vessel,etc.). In the case of hemophilia or other bloodcoagulation/clotting/bleeding disorders, an improvement can reduce thefrequency or duration of bleeding episodes or hemorrhage. Improvementslikewise include chronic disorders associated with bloodcoagulation/clotting/bleeding associated disorders such as a reductionin neurological problems, crippling tissue and joint damage, forexample.

The methods of the invention for treating a subject are applicable forprophylaxis to prevent a condition in a subject, such as a hyperglycemiccondition or an associated disorder, or development of obesity or anincreased body mass. Alternatively, the methods can be practicedfollowing treatment of a subject as described herein. For example,following treatment and a reduction of body mass to the desired weight,leptin, GLP-1, PYY or CCK can be periodically produced by gut mucosalcells, as described herein, in order to suppress appetite, decrease mealconsumption, etc. thereby maintaining desired body weight.

The methods of the invention for treating a subject also can besupplemented with other forms of therapy. Supplementary therapiesinclude drug treatment, a change in diet (low sugar, fats, etc.)surgical resection, transplantation, radiotherapy, etc. For example, amethod of the invention for treating a hyperglycemic condition can beused in combination with drugs or other pharmaceutical formulations thatincrease insulin or lower glucose in a subject. Drugs for treatingdiabetes include, for example, biguanides and sulphonylureas (e.g.,tolbutamide, chlorpropamide, acetohexamide, tolazamide, glibenclamideand glipizide). Appetite suppression drugs are also well known and canbe used in combination with the methods of the invention. Supplementarytherapies can be administered prior to, contemporaneously with orfollowing the invention methods of treatment. The skilled artisan canreadily ascertain therapies that may be used in a regimen in combinationwith the treatment methods of the invention.

Pharmaceutical Formulations

As the methods of the invention can include contacting a mucosal cell(s)present in a subject with a polynucleotide, the present invention alsoprovides “pharmaceutically acceptable” or “physiologically acceptable”formulations comprising chitosan-based nanoparticles of the invention.Such formulations can be administered in vivo to a subject in order topractice the treatment methods of the invention.

As used herein, the terms “pharmaceutically acceptable” and“physiologically acceptable” refer to carriers, diluents, excipients andthe like that can be administered to a subject, preferably withoutproducing excessive adverse side-effects (e.g., nausea, abdominal pain,headaches, etc.). Such preparations for administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions.

Pharmaceutical formulations can be made from carriers, diluents,excipients, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with administration to a subject. Such formulations canbe contained in a tablet (coated or uncoated), capsule (hard or soft),microbead, emulsion, powder, granule, crystal, suspension, syrup orelixir. Supplementary active compounds and preservatives, among otheradditives, may also be present, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.

A pharmaceutical formulation can be formulated to be compatible with itsintended route of administration. The preferred route of administrationin the present invention is oral, endoscopic, or rectal. Thus, preferredpharmaceutical formulations include carriers, diluents, or excipientssuitable for administration by routes including oral, endoscopic andrectal, though other formulations and other routes of administrationcapable of reaching the gut lumen, especially of the stomach, smallintestine, and colon are contemplated.

For oral administration, a composition can be incorporated withexcipients and used in the form of tablets, troches, or capsules, e.g.,gelatin capsules. Pharmaceutically compatible binding agents, and/oradjuvant materials can be included in oral formulations. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or flavoring.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. For example, a time delay material such as glycerylmonostearate or glyceryl stearate alone, or in combination with a wax,may be employed.

Suppositories and other rectally administrable formulations (e.g., thoseadministrable by enema) are also contemplated. Further regarding rectaldelivery, see, for example, Song et al., Mucosal drug delivery:membranes, methodologies, and applications, Crit. Rev. Ther. Drug.Carrier Syst., 21:195-256, 2004; Wearley, Recent progress in protein andpeptide delivery by noninvasive routes, Crit. Rev. Ther. Drug. CarrierSyst., 8:331-394, 1991.

Additional pharmaceutical formulations appropriate for administrationare known in the art and are applicable in the methods and compositionsof the invention (see, e.g., Remington's Pharmaceutical Sciences (1990)18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12thed., Merck Publishing Group, Whitehouse, N.J.; and PharmaceuticalPrinciples of Solid Dosage Forms, Technonic Publishing Co., Inc.,Lancaster, Pa., (1993)).

Administration

A preferred route of administration is oral. For oral administration, acomposition can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included inoral formulations. The tablets, pills, capsules, troches and the likecan contain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or flavoring.

Endoscopes, cannulas, intubation tubes, catheters and the like can beused to deliver the formulation to various parts of the gut of asubject. This allows effective delivery and targeting of nanoparticlesto particular areas of the gut. In one aspect, the invention providesdelivery devices comprising a chitosan-based nanoparticle compositiondisclosed herein.

The doses or “effective amount” for treating a subject are preferablysufficient to ameliorate one, several or all of the symptoms of thecondition, to a measurable or detectable extent, although preventing orinhibiting a progression or worsening of the disorder or condition, or asymptom, is a satisfactory outcome. Thus, in the case of a condition ordisorder treatable by expressing a therapeutic nucleic acid in gutmucosal cells, the amount of therapeutic RNA or therapeutic proteinproduced to ameliorate a condition treatable by a method of theinvention will depend on the condition and the desired outcome and canbe readily ascertained by the skilled artisan. Appropriate amounts willdepend upon the condition treated, the therapeutic effect desired, aswell as the individual subject (e.g., the bioavailability within thesubject, gender, age, etc.).

Veterinary applications are also contemplated by the present invention.Accordingly, in one embodiment, the invention provides methods oftreating non-human mammals, which involve administering a chitosan-basednanoparticle of the invention to a non-human mammal in need oftreatment.

The effective amount can be ascertained by measuring relevantphysiological effects. For example, in the case of diabetes or otherhyperglycemic condition, a decrease in blood glucose or an improvementin glucose tolerance test can be used to determine whether the amount ofinsulin is effective to treat the hyperglycemic condition. For example,an amount reducing FPG from 126 mg/dl to 120, 115, 110, or less is aneffective amount. In the case of obesity or an undesirable body mass, adecrease in the subjects' mass, a decrease in meal size or caloriccontent of a meal, increased satiety for a given meal size, anddecreases in serum/plasma levels of lipid, cholesterol, fatty acids, LDLor VLDL all can be effective amounts for ameliorating obesity or anundesirable body mass of a subject. In the case of hemophilia, aneffective amount is an amount which reduces clotting time or frequencyor duration of bleeding episodes in a subject.

The mucous of the mucosal tissue may be removed or otherwise preparedprior to administration, for example, using penetrants or other barrierpenetration enhancers. Such penetrants appropriate to the barrier to bepermeated are generally known in the art, and include, for example, fortransmucosal administration, incubation with N-acetyl-cysteine(Nakanishi et al. Chem Pharm Bull (Tokyo) 40:1252 (1992), Meaney andO'Driscoll Eur J Pharm Sci. 8:167 (1999); hydrolysis of intestinalmucins by purified Sigma 1 protein and infectious subviral particles(Bisaillon et al. J Mol. Biol. 286:759 (1999); removal of mucous andincrease in gene transfer by Dodecyl β-D-maltoside Connor et al, GeneTher. 2001 January; 8(1):41-8; desialation (Slomiany et al. GenPharmacol. 27:761 (1996); (Hirmo et al. FEMS Immunol Med. Microbiol.20:275 (1998); desulphation by H. pylori glycosulfatase (Slomiany et al.Am J GastroenteroL 87:1132 (1992); desialation by neuraminidase (Hanskiet al. Cancer Res. 51:5342 (1991)); disulphide bond breakage byβ-mercaptoethanol (Gwozdzinski et al. Biochem Int. 17:907 (1988);deglycosylation with specific exoglycosidases such as fucosidase,β-galactosidase, N-acetyl-galactosaminidase, β-N-acetylhexososaminidase, and neuraminidase (Slomiany et al. Biochem Biophys ResCommun. 142:783 (1987); acid removal of by 0.4 N HCl (Ruggieri et al.Urol Res. 12:199 (1984), Davis C. P. and Avots-Avotins A. E. ScanElectron Microsc. (Pt 2):825-30 (1982), Parsons et. al. Am J. Pathol.93:423 (1978)), among others.

The number of precursor cells in the gut mucosa can be increased byexposure to cytotoxic agents and growth factors. For example,irradiation of the small gut increases the number clonogenic/stem cells(Roberts S. A. Radiat. Res. 141:303 (1995); Cai W. B. et. al. Intl. J.Radiat. Biol. 71:145 (1997)). In addition, treatment with GLP-2,epidermal growth factor, TGF-α, insulin-like growth factors,interleukins, among others, have been shown to promote the growth ofmucosal cells (Potten C. S. Int. J. Exp. Path 78:219 (1997)). In thisway, additional target cells can be produced thereby increasingtransfection efficiency and subsequent regulated protein production bymodified gut mucosal cells.

Physicochemical Properties of Various Chitosan/DNA Polyplexes

Table 4 shows physiochemical properties of exemplary chitosan-basednanoparticles.

Avg. No. Monomers mol. Particle Zeta based on wt DDA N:P size potentialChitosan Avg. Weight kDa (%) ratio (nm) (mV) P3; C(15, 98) 15 2.4 98 60140 +34.8 P1; C(24, 98) 24 3.9 98 60 123 +37.9 RCO5; 1199 206 74 2.9 205+6.2 C(1199, 74) CH01; 2038 332 95 3.82 243 +8.5 C(2038, 95) CH10; 2036342 83 3.24 243 +6.5 C(2036, 83)

Size was measured at 30 minutes and zeta potential was measured at 5hours, each composition having a DNA concentration of 50 μg/ml, and a pHof 5.5 (RC05, CH01, CH10) or 5.0 (P1 and P3). Using the “C” nomenclaturefor chitosan, the first factor refers to the average number of monomers,and the second factor refers to the DDA.

As DNA concentration is varied in compositions composed of chitosanpolymers having the same or similar average molecular weight, the N:Pratio is preferably varied for optimization. For example, forcompositions comprising chitosan-based nanoparticles composed ofchitosan polymers having an average molecular weight of about 3.9 kDaand a DDA of about 98% (P1 above), and a DNA concentration of about 50μg/ml, a highly preferred N:P ratio is 60:1. For a compositioncomprising the same chitosan-based nanoparticles composed of chitosanpolymers having an average molecular weight of about 3.9 kDa and a DDAof about 98%, but a DNA concentration of about 250 μg/ml, a more highlypreferred N:P ratio is 20:1. In general, as the DNA concentration isincreased, it is preferable to decrease the N:P ratio, especially forlow molecular weight chitosan nanoparticles.

At pHs below the pKa of the amine group of chitosan, the N:P ratio moreclosely reflects the actual charge ratio in the nanoparticle. In apreferred embodiment, the compositions of the invention have a pH ofless than 6.5, more preferably less than 6.0, and most preferablybetween about 4.5 and about 5.5, and N:P ratio very closely reflectsactual charge ratio.

Notably, zeta potential varies with pH and the extent of protonation ofchitosan amine groups. Generally, as the pH increases and the extent ofprotonation of chitosan amine groups decreases, the zeta potentialdecreases.

Also, as the degree of acetylation (DDA) is varied for chitosan polymershaving the same average monomer length, it is preferable to adjust theN:P ratio in compositions. For example, for compositions comprisingchitosan-based nanoparticles having an average monomer length of about24, a DDA of about 98%, and a DNA concentration of about 250 μg/ml, ahighly preferred N:P ratio is 20:1. For a composition comprising thesame chitosan-based nanoparticles having an average monomer length ofabout 24, a DNA concentration of about 250 μg/ml, but a DDA of about80%, a highly preferred N:P ratio is 40:1. In general, as the DDA isdecreased, it is preferable to increase the N:P ratio, especially forlow molecular weight chitosan nanoparticles.

All citations are expressly incorporated herein in their entirety byreference.

Experimental

Formation of Chitosan-Based Nanoparticles

(compositions relating to data FIGS. 1-10) Plasmid DNA and chitosansolutions, prepared separately, were adjusted to a concentration equalto two-times the required final concentration. For P3 and P1, DNA wasdiluted in water and the indicated chitosan was dissolved in 5 mM sodiumacetate, pH5.0. For other complexes in Table 5, DNA was diluted in 50 mMsodium sulfate solution, and the indicated chitosan was dissolved in 5mM sodium acetate, pH5.5. In all cases, both solutions were incubated at55° C. for 5 minutes before being mixing. Equal volumes of the twosolutions were mixed and rapidly vortexed for 30 seconds to formDNA/chitosan particles. For certain applications, this preparation wasfurther diluted in various buffers prior to analysis. Chitosan used wasobtained from FMC (Norway) and Biosyntech (Canada).

TABLE 5 Chitosan-based Nanoparticle Formulations avg # In vitro - Gelelectrophoresis analysis monomer % N:P ratios W:W ratios DNAConcentration Polymer units Deacetylation tested (chi:DNA) tested(ug/ml) P3 15 98 60 31 50 50 26 50 40 21 50 30 15.5 50 15 7.8 50 7.5 3.950 3.75 1.9 50 2 1 50 1 0.5 50 P1 24 98 120 60 50 80 40 50 60 30 50 5025 50 40 20 50 30 15 50 25 12.5 50 20 10 50 15 7.5 50 10 5 50 7.5 3.7550 3.75 1.875 50 2 1 50 1 0.5 50 AS-111-39-A 98 84 30 18.5 50 15 9.25 507.5 1.23 50 1 0.62 50 AS-111-39-B 260 84 20 12.3 50 10 6.2 50 5 3.1 50 10.62 50 UPC113 677 86 3.40 2 50 RC05 1199 74 5.7 4 25, 50 2.85 2 50, 2001.90 1.33 75 1.42 1 50, 100 0.95 0.665 150 0.71 0.5 200 CH01 2038 953.82 2 50 1.91 1 100 0.96 0.5 200 0.38 0.2 500 CH10 2036 83 3.24 2 50,200 1.62 1 100 0.33 0.2 500

Transfection of 293T Cells Using Chitosan-Based Nanoparticles

Human embryonic kidney cells carrying the SV40 large-T antigen (293T)were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with10% fetal calf serum. They were trypsinized, washed in PBS and plated incomplete medium at approximately 6×10⁵ cells in 3 ml of media per wellof a 6-well plate. For DNA/chitosan-mediated transfection, cells werewashed the following day with serum free Optimem prior to addition ofthe DNA/chitosan particles. The medium used for these incubations waseither serum-free Optimem, pH 7.4 or Optimem adjusted to pH 5.0. 4 hoursafter transfection, 3 ml of Optimem, pH 7.4 was added to each well.Forty-eight hours after transfection with a β-galactosidase plasmidunder control of the CMV promoter (pShuttle-CMV-LacZ), cells wereassayed for β-galactosidase activity using the Galacto-Light Plus System(ABI). Briefly, cells were washed twice with PBS, lysed with 200 μllysis buffer containing protease inhibitor and detached with a cellscraper. Cell lysate was clarified by spinning at maximum speed for 2min and 20 ml was assayed according to manufacturer's instructions.Signal was detected using the LMax II384 luminometer (Molecular Devices)with a 1 sec integration. The β-galactosidase activity was corrected fortotal protein in the sample by measuring protein content using theBio-Rad DC Protein assay according to the manufacturer's instructions.

Nanoparticle Formation Analysis

The ability of chitosan to complex and retain its interaction with DNAwas assessed by gel electrophoresis in 0.8% agarose in 0.04M Tris,0.001M sodium acetate, 0.02M EDTA buffer, pH 8 (TAE) with ethidiumbromide (0.2 μg/ml). Gels were run at 100 mV for 1 h and DNA retentionwas visualized under UV light. (data not shown)

Viral Vectors

Gene transfer vectors based on feline immunodeficiency virus (FIV) andadeno-associated virus (AAV) carrying marker genes of interest (e.g.insulin, β-galactosidase or SEAP) were produced. FIV vector wasgenerated using standard methods. AAV2 particles carrying transgenes ofinterest were also generated using standard methods. Particles wereharvested by using a HiTrap Heparin HP column (Amersham) three daysfollowing transfection.

Gene Transfer to the Duodenum In Vivo

Chitosans CH01 (N:P 3.82), CH10 (N:P 3.24), RC05 (N:P 2.85), P1 (N:P 60)and P3 (N:P 60) were used to form complexes with 12.5 μg plasmid DNA (50μg/ml), as described above, prior to administration to mice (FIG. 2).Additional experiments using P1 and RC05 used a range of N:P ratios asdescribed in FIG. 3. Briefly, an abdominal incision was made inanaesthetized and overnight-fasted mice. A section of the duodenum (2 cmfrom the pyloric sphincter) was isolated and externalized with animmobilized glass hook. The lumen of the duodenal section was washedonce with saline and optionally incubated with a mucolytic agent (10%NAC) for 10 minutes. Following the incubation, the mucolytic agent waspushed to the distal region of the intestinal tract and washed thricewith saline. Two hundred microlitres of the gene transfer solution(chitosan-based nanoparticle preparation, or comparative viral vector)was then delivered into the lumen of the isolated duodenal section andincubated for 1 hour with the duodenal section stabilized with a glasshook in an elevated position. The externalized gut section was coveredin a saline-soaked gauze to prevent excessive tissue drying. Followingincubation, the duodenal section was returned to the abdomen and theincision was closed with 5-0 vicryl suture. Each mouse was administered100 mg/kg ampicillin (i.p.), 5 mg/kg ketaprofen (s.c.) and 1 ml lactatedringers solution (s.c.) upon completion of the surgery. Animals werethen ear marked and returned to their cages to recover on heating pads.

For oral administration of DNA/chitosan complexes, 500 μl of testsolution was administered to the stomach of mice using a gavage needle.

Quantitative PCR to Measure Gene Delivery to the Duodenum

The amount of DNA transfected into duodenal tissue was measured byquantitative polymerase chain reaction (Q-PCR). Tissue samples werethawed to room temperature then ground with a pestle and mortar. DNA wasextracted using the manufacturer's instructions for the Qiagen DNeasyTissue Kit, except that double the listed volumes of proteinase K andATL buffers were used. Genomic DNA concentrations were measured using aspectrophotometer prior to amplification using primers specific for theparticular transgene being tested. Standardization was also carried outusing primers for 18S RNA coding sequence. The amplification wasquantified using blank samples spiked with plasmid DNA. Amplificationwas carried out using and ABI 7000 cycler.

Long-Term Increase in Systemic Protein Following Transfection of GutMucosal Cells

The ability to generate long-term expression was assessed in the smallintestine of mice. The SEAP plasmid complexed with chitosan was appliedto the duodenum as described above. The plasmid contained the SEAP geneunder the control of the constitutive promoter EF1α. The plasmid alsocontained a bacterial origin of replication and an ampicillin selectablemarker.rGIP-hlNS.

Integration of Therapeutic Nucleic Acid

Chitosan P1 (N:P 60) used to form complexes with a total of 12.5 μgplasmid DNA (50 μg/ml) prior to administration to mice, as describedabove. Reporter gene integration plasmid contains an origin ofreplication, an ampicillin resistance gene and a CMV promoter drivingthe expression of the beta-galactosidase reporter gene (pMM2611). Thereporter cassette is flanked on either side by inverted terminal repeats(ITRs) which are recognized and integrated into the host genome by themariner transposase. The mariner transposase itself was delivered on thepCMV-9 plasmid, driven by the CMV promoter, which also contains anorigin of replication and an ampicillin resistance gene used forselection during bacterial propagation. See FIG. 7 for plasmid maps. Theplasmids were mixed at a 5:1 (pMM2611:pCMV-C9) prior to complexationwith chitosan. FIG. 6 shows cotransfection of mouse duodenum cells invivo with chitosan-based nanoparticles P1 at a NP ratio of 60:1.

Measurement of SEAP Activity In Vivo

SEAP levels in mouse plasma were determined using the Rochechemiluminescent SEAP Reporter Gene Assay. Frozen samples were thawed,vortexed, spun down, diluted and heat inactivated at 69° C. for 45 min.After heat inactivation, samples were cooled on ice, centrifuged andassayed according to manufacturer's instructions. Signal was detectedusing the LMax 11384 luminometer (Molecular Devices) with a 1 secintegration.

Results: FIG. 1 shows results of in vitro transfection of 293 cells withchitosan-based nanoparticles comprising chitosan polymers of variousmolecular weights and degrees of deacetylation (see Table 4 fordetails). Chitosans CH01 (N:P 7.6), CH10 (N:P 6.5), RC05 (N:P 5.7), P1(N:P 60) and P3 (N:P 60) were complexed with the pShuttle-CMV-LacZplasmid prior to transfection and β-galactosidase activity was measuredas a measure of transfection efficiency. These results show differencesin transfection efficiency depending on the particular chitosan used andvery significant levels of transfection relative to naked DNA.

FIG. 2 shows results of in vivo transfection of murine luminal cells ofthe duodenum with chitosan-based nanoparticles comprising chitosanpolymers of various molecular weights and degrees of deacetylation (seeTable 4 for details). Chitosans CH01 (N:P 3.82), CH10 (N:P 3.24), RC05(N:P 2.85), P1 (N:P 60) and P3 (N:P 60) were complexed with thepShuttle-CMV-LacZ plasmid prior to transfection, and β-galactosidaseactivity was measured as a measure of transfection efficiency. Theseresults show differences in transfection efficiency depending on theparticular chitosan used. RC05 also gives a clear illustration ofdifferences between the transfection efficiency obtained in vitro and invivo.

FIG. 3 shows results of in vivo transfection of murine luminal cells ofthe duodenum with chitosan-based nanoparticles comprising chitosanpolymers of various molecular weights (see Table 4 for details) and atvarious N:P ratios, as indicated. The pShuttle-CMV-LacZ plasmid was usedfor transfection, and β-galactosidase activity was measured as a measureof transfection efficiency. These results show differences intransfection efficiency depending on the NP ratio used and that theoptimal NP ratio is different for various chitosan molecules.

FIG. 4 shows results of in vivo transfection of murine luminal cells ofthe duodenum with RC05 chitosan-based nanoparticles at an N:P ratio of2.85:1, compared to transduction with FIV (109 transduction units) andAAV (1011 transduction units) particles. Data represent the number ofgene copies detected in the tissue using quantitative PCR. This datashows that chitosan-mediated gene transfer to the gut using particularformulations is more effective than what are normally consideredeffective viral gene transfer systems.

FIG. 5 shows results of in vivo transfection (single administration) ofmurine luminal cells of the duodenum with P1 chitosan-basednanoparticles at an N:P ratio of 60:1, wherein the nanoparticlecomprises an EF1a-SEAP plasmid. The level of SEAP protein in blood atvarious time points is shown. This data illustrates the ability ofchitosan to deliver long-term gene expression in the duodenum of mice.

FIG. 6 results of in vivo transfection and effect of integration inmurine luminal cells of the duodenum with P1 chitosan-basednanoparticles at an N:P ratio of 60:1. Two plasmids (pMM2611-beta-galcontaining one ITR on either side of the LacZ gene; pCMV-C9 encoding theMariner integrase) were mixed at a ratio of 5:1 prior to complexationwith P1 chitosan. Duodenum was harvested 14 days following transfectionand the presence of pMM2611-beta-gal plasmid was measured byquantitative PCR.

FIG. 7 shows two schematics of the plasmids, pCMV-C9 (Panel A) andpMM2611-beta-gal (Panel B), used in the experiment shown in FIG. 6. Thediagrams show the main sequence elements such as promoters, transgenesand ITR integration sites. The principal restriction enzyme sites arealso shown.

Chitosan Mediated Gene Transfer to Duodenal Mucosal Cells in Mice at 2Days Post Delivery, and Persistence at 14 Days Post Delivery with Use ofφC31

Q-PCR on mouse duodenum after gene transfer with chitosan-DNAnanoparticles: To quantitate gene copy number after luminal delivery ofchitosan-DNA nanoparticles to the duodenum of mice, mouse duodenum wascollected 2 or 14 days post polyplex delivery and the DNA was extractedusing the Qiagen DNeasy Tissue Kit. Real-time quantitative PCR (Q-PCR)was subsequently performed on 1 μg of DNA with TaqMan PCR Master Mix(Applied Biosystems) and primers and probes specific to either the LacZor luciferase gene. Q-PCR using 18S primers and probe (AppliedBiosystems) was performed as an internal control.

To confirm the efficiency of gene transfer using chitosan-basednanoparticles, we delivered naked DNA (pShuttle-CMV-LacZ) orpShuttle-CMV-LacZ packaged with C(24,98) to the duodenum of mice. After2 days, we found that the gene copy numbers for the group that receivedchitosan-DNA nanoparticles was more than 100-fold higher than the groupthat received naked DNA (FIG. 8, left). This confirms the ability ofchitosan-based nanoparticles to effectively deliver DNA to the cells ofthe duodenum.

A plasmid containing an expression cassette of a luciferase marker geneand attB sequence (pLuc-attB) was packaged with C(24,98) with or withoutthe ΦC31 integrase expression plasmid (pCMV-INT) and delivered to theduodenal lumen of mice. When a ΦC31 integrase expression plasmid wasco-packaged in the nanoparticles, the presence of the marker genepersisted in the gut mucosa at 14 days post-delivery at a level that wasover 100 fold higher than in mice treated with nanoparticles carryingthe pLuc-attB plasmid alone. The significantly higher levels ofluciferase gene in the gut at 14 days post vector delivery indicatesthat the ΦC31 integrase successfully integrated the transgene into along-living duodenal mucosal precursor cell population.

C-Peptide Levels in Mouse Plasma after Delivery of C(24,98)-GIP-INS orC(1119,74)-GIP-INS

Polyplex Formation: Plasmid DNA and chitosan solutions, preparedseparately, were adjusted to a concentration equal to two-times therequired final concentration. For P1 (C(24,98)), DNA was diluted inwater and the indicated chitosan was dissolved in 5 mM sodium acetate,pH5.0. For RC05 (C(1119,74)), DNA was diluted in 50 mM sodium sulfatesolution, and the indicated chitosan was dissolved in 5 mM sodiumacetate, pH5.5. In each case, both solutions were incubated at 55° C.for 5 minutes before being mixing. Equal volumes of the two solutionswere mixed and rapidly vortexed for 30 seconds to form DNA/chitosanparticles. Chitosan used was obtained from FMC (Norway) and Biosyntech(Canada).

Plasma collection: Mouse plasma was collected from the saphenous vein ofmice at various time points after luminal delivery of C(24,98)-GIP-INSor C(1119,74)-GIP-INS (total DNA dose=10 μg/animal). The mice werefasted overnight and then given 2 g of glucose per kg of mouse 15minutes prior to the blood collection. Human C-peptide levels in mouseplasma were determined by using the ALPCO Ultra-Sensitive Insulin ELISAkit following the manufacturer's recommendations, with minormodifications. These modifications included: mouse plasma diluted 1:2instead of using neat and incubated in the assay buffer for 2 h insteadof 1 h.

Results (FIG. 9): To evaluate the potential therapeutic efficacy ofnanoparticles in targeting insulin production to gut K-cells, wepackaged the GIP/Ins transgene with attB sequence(prnGIP-hlNS-WPRE-attB) and ΦC31 integrase expression plasmid (pCMV-INT)(INS:INT ratio=5:1) with chitosan (either C(24,98) NP=60 or C(1119,74)NP=2.9) and delivered the polyplex to the duodenum of mice, using theluminal delivery method described above. We were able to achievedetectable circulating levels of human C-peptide in mice for over 120days. Following a single vector administration with C(24,98) polyplex tothe duodenum, the mean levels of plasma human C-peptide obtained inthese mice reached as high as 10 pM after about 80 days following vectordelivery. In contrast, animals that received C(1119,74)-GIP-INS had aninitial early increase in circulating C-peptide levels, followed bysustained low levels and loss of C-peptide by 100 days. This study showsthat low molecular weight chitosan nanoparticles are more effective atachieving robust insulin production and secretion by K cells for periodslonger than 50 days.

(FIG. 10) To determine if in vivo human insulin production and secretionfrom K-cells is meal-regulated, overnight fasted mice were given 0.5 gstandard chow pellet that was consumed entirely in ˜8 minutes or 2 g/kgbody weight of dextrose via oral gavage. Thirty minutes after theintroduction of food or fifteen minutes after introduction of dextrose,plasma human C-peptide in treated animals increased significantly,from >2 pM fasted level to ˜4.8-6 μM post introduction of dextrose orstandard chow. 60 minutes post oral gavage of dextrose solution, thehuman C-peptide level return to basal level. These results indicate thatinsulin secretion from gut K-cells is meal-regulated; and like b-cells,they are capable of secreting insulin rapidly in response to a meal.

C-Peptide Levels in Mouse Plasma after Delivery of VariousChitosan-GIP-INS Particles

Polyplex Formation (data FIGS. 11-14): Chitosan powder was added to 0.5%aqueous solution of acetic acid until the chitosan working solutionreaches pH 4.8. The working solution is then filtered through a membranefilter (Acrodisc 0.2 μm pore size, Pall Life Sciences). Stock DNAsolutions (in 1×TE) of plasmid A and plasmid B were mixed in a 5:1 ratioof plasmid A to plasmid B, diluted in water, and then filtered through amembrane filter (Acrodisc 0.2 μm pore size, Pall Life Sciences) toproduce the DNA working solution. The concentrations of the workingsolutions were varied in order to produce a final product with thedesired DNA and chitosan concentrations according to Table 6.DNA-Chitosan polyplexes were manufactured by continuous in-line mixingof chitosan and DNA working solutions at a volume ratio of 2:1.

TABLE 6 Required working solution concentrations for different polyplexformulations Concentration in Concentration in Target Mixed PolyplexWorking Solutions Chitosan mer NP DNA Chitosan DNA Chitosan and DDARatio (mg/mL) (mg/mL) (mg/mL) (mg/mL) C(24, 98) 20 0.075 0.75 0.11252.25 C(24, 98) 40 0.075 1.5 0.1125 4.5 C(24, 98) 60 0.075 2.25 0.11256.75 C(65, 98) 5 0.075 0.1875 0.1125 0.5625 C(65, 98) 20 0.075 0.750.1125 2.25 C(65, 98) 30 0.075 1.125 0.1125 3.375 C(24, 98) 20 0.25 2.50.375 7.5 C(65, 98) 5 0.25 0.625 0.375 1.875 C(165, 98) 2 0.25 0.250.375 0.75 C(58, 80) 5 0.25 0.802 0.375 2.406

Polyplex delivery to mice was done as described above.

Mouse plasma collection: Mouse plasma was collected from the saphenousvein of mice at various time points after luminal delivery ofC(24,98)-GIP-INS, C(65,98), C(165,98) or C(58,80) (total DNA dose=50μg/animal).). The mice were fasted overnight and then given 2 g ofglucose per kg of mouse 15 minutes prior to the blood collection. HumanC-peptide levels the mouse plasma were determined by using the ALPCOUltra-Sensitive Insulin ELISA kit following the manufacturer'srecommendations, with minor modifications. These modifications included:mouse plasma diluted 1:2 instead of using neat and incubated in theassay buffer for 2 h instead of 1 h.

Results (FIG. 11): We performed a study to compare the efficiency oftargeted insulin production in the K cells of the mice with variouschitosan polymers varying in chain length and percentage deacetylation(DDA). Chiosan polyplex were formulated to contain the human insulingene linked to the rat GIP promoter (prnGIP-hlNS-WPRE-attB) and the ΦC31integrase expression plasmid (pCMV-INT) (INS:INT ratio=5:1). We foundthat C(24,98) resulted in the highest levels of human C-peptideproduction starting 5 days after administration and that these levelswere sustained throughout the study duration of 109 days. Longer chainpolymers with the same DDA, C(65,98) and C(165,98), initially producedsignificant but lower levels of human C-peptide, which declined to belowthe detection limit by 50 days. Use of nanoparticles comprised ofintermediate length polymers having a lower degree of deacetylation(DDA) did not result in significant human C-peptide levels at the givenN:P ratio and DNA concentration. This study shows that low molecularweight chitosan nanoparticles are more effective at achieving robustinsulin production and secretion by K cells for periods longer than 50days. Further, this study and the results shown above in FIG. 9 supportthat in addition to providing for a longer duration of expression, lowmolecular weight chitosan nanoparticles provide for higher levels ofexpression. Without being bound by theory, this appears to reflect theability of low molecular weight chitosan nanoparticles to transfectgreater numbers of precursor cells, resulting in a greater numbers ofdescendant cells (including K cells) having an integrated GIP-insulintransgene.

Human GIP Promoter-Driven Expression of Human Insulin in Pig Duoedenum

Chitosan/DNA polyplex preparation was done as described above (FIG. 11).The FIV vector plasmid carrying the rat GIP promoter linked to humaninsulin transgene (FIV-rGIP/hlns) was generated with thewell-established three-plasmid transfection system. To package therGIP/hlns transgene into VSV-G pseudotyped FIV viral particles, 293Tcells are transfected with vector plasmids pFLX-rGIP/hlns, packagingplasmid (pCPRΔEnv) and pCMV-VSV-G for pseudotyping. In detail, 293Tcells are passed into thirty 150 mm tissue culture dishes the day beforetransfection. A total of 20 ug of DNA (8 ug of pFLX-rGIP/hlns, 8 ug ofpCPRΔEnv and 4 ug of pCMV-VSV-G) are transfected into 80% confluentcells using calcium phosphate precipitation method. Eight hours aftertransfection, cells are fed with fresh Dulbecco's Modified Eagle Media(D-MEM) containing 10% FCS and incubated at 37° C. overnight. Theculture media is replaced the next morning and the transfected cells aretransferred to a 32° C. incubator. Viral particles are harvested fromthe culture media at 48 h and 72 h post transfection. The supernatantcontaining the viral particles is clarified by low speed centrifugation,filtered through 0.45 mm filters and subsequently concentrated bycentrifugation at 38,000 g for 2.5 h at 4° C. The pellet is thenre-suspended in TNE buffer (50 mM of Tris and 130 mM of NaCl and 1 mM ofEDTA). Concentrated FIV vector is stored at −80° C. For each batch ofviral vector produced, vector titers are determined by real-time PCRusing probes specific for the human insulin.

Yorkshire and Yucatan pigs were obtained through the University ofBritish Columbia Animal Care Centre (ACC) under enGene's UBC ProtocolA03-0101 (Gene Transfer to Intestinal Cells of Mammals). Study protocolswere reviewed and approved independently by the Animal Care andBiohazard Committees at UBC. Animals were housed at the ACC andtransported to the Animal Resource Centre (ARU) where endoscopicprocedures and tissue collection were performed.

Pigs were fasted overnight and pre-medicated by intramuscular injectionof a mixture of atropine (0.04-1.00 mg/kg), ketamine hydrochloride(10-20 mg/kg), and rompun (0.1 mg/kg) by ARU staff. Subsequently,endotracheal intubation was performed on the animal for the delivery of2% isoflurane, which was maintained throughout the entire procedure.Ringer's lactate was administered by intravenous catheterization forvolume replacement. Pigs weighed between 29 and 51 kg at the time ofvector delivery.

Pigs were placed in a dorsal recumbency with the abdominal regioncleaned with hibi-cleanse, sanitized with alcohol and sterilized withiodine. A midline incision was made using cautery through the skin fromthe level of the zyphoid process moving distal to the inguinal region.The same incision was repeated through the underlying muscle layers andthe linea alba using a 10 blade scalpel. Four laparotomy towels wereplaced along the midline to allow for placement of an abdominalretractor. The pyloric sphincter and proximal duodenum were isolated andsix sections were marked off with superficial circular suture patternsusing 4.0 vicryl. Superficial injections (2.5 ml per injection site)were made into the mucosa within the marked off treatment site.

Seven days after delivery, pigs were pre-medicated and anesthetized asdescribed above. The abdomen was surgically opened as described above tolocate the marked region of the duodenum. Clamps were placed proximaland distal to the treatment site to clamp off the duodenum. Bloodvessels adjacent to the duodenum were clamped and then severed. Theclamped duodenal sections were incised and the segment removed from theabdomen and placed into ice-cold saline for washing. Following clampremoval, the duodenum was cut open longitudinally, washed with freshice-cold saline and pinned to a cutting board. A scalpel was used todissect the marked regions of duodenum with a 5 mm outer margin toensure all treated tissue is harvested. Each of these sections was thencut into 2 for both RT-Q-PCR and IHC. Tissue samples for RT-qPCR weresubmerged in TRIzol reagent (Invitrogen Canada Inc; Burlington, ON),then snap-frozen in dry ice. Tissue samples for IHC were harvested andfixed in the 4% parafolmaldehyde.

Real-time quantitative RT-PCR(RT-q-PCR): Tissues in TRIzol reagent washomogenized using a ultra-turrax T8 homogenizer (IKA Works Inc.;Wilmington, N.C.). Total tissue RNA was isolated and purified accordingto the manufacture's protocol.

Each RNA sample was first digested by DNase I (Invitrogen) to remove anyDNA contamination. To each 1 ug sample of RNA, 1 μL 10× DNase I ReactionBuffer, 1 μL DNase I and RNase/DNase free water were added to a finalvolume of 10 μL. The tubes were incubated for 15 minutes at RT and thenthe DNase I was inactivated by addition of 1 μL of 25 mM EDTA. Thesamples were heated for 10 minutes at 65° C.

Reverse Transcription: First-Strand cDNA was synthesized by usingSuperScript™ II Reverse Transcriptase (Invitrogen). Following DNAdigestion, 1 μL each of Random Primer (Invitrogen) and 10 mM dNTP mix(Invitrogen) were added to each tube, which was heated 65° C. for 5minutes, then put on ice. To each tube, the following were added: 4 μLof 5× first-Strand Buffer (Invitrogen), 2 μL of 0.1M DTT, and 1 μL ofRNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen), and the tubeswere incubated at 42° C. for 2 minutes. 1 μL of SuperScript™ II was thenadded and the tubes were incubated at 42° C. for 50 minutes. Finally,the reaction was terminated by heating at 70° C. for 15 minutes.

Real-Time PCR: A pair of PCR primers and a probe were designed toamplify a portion of the human insulin gene and 18S primers were used tonormalize the cDNA quantity of 18S and human insulin between tissuesfrom the experimental and control groups. The sequences of the humaninsulin primers: 5′ CGCCCTTGCTGCATCAG 3′ (Forward), 5′ GCAGGAGGCGCATCCA3′ (Reverse) (IDT Inc.; Coralville, Iowa). The insulin probe used was6FAM CTCACACCTGGTGGAAG MGBNFQ (Applied Biosystems, Foster City, Calif.).18S primer and probe, TaqMan Master Mix was also purchased from AppliedBiosystems. All samples were tested for both insulin gene and 18S.Working in a lamina flow hood, duplicate samples were loaded into aMicroAmp® optical 96-well reaction plate (Applied Biosystems). A 10 μLsample of cDNA (contained 0.3 ug of original RNA), 0.25 uL of probe,12.5 μL TaqMan Master Mix, 1.125 μL of each insulin primer or 10 uL ofsample cDNA (contained 0.5 ng of original RNA), 1.25 uL of 18S primerand probe and 1.25 μL RNase/DNase free water, and 12.5 uL of TaqManMaster Mix were added to each well. All samples were run in a ABI PRISM7000 Sequence Detection System (Applied Biosystems). The data werenormalized by the 18S and the relative quantitative difference betweenexperimental group and control animals was expressed by the Log 2 FoldChange.

Immunohistochemistry (data not shown): Pig duodenum samples were fixedin 4% paraformaldehyde in 0.1 mol/liter PBC (pH 7.5) for 4 h and washedin 70% ethanol. Paraffin-embedded sections (5 um) were incubated in 10mM sodium citrate (pH 6.0) solution for 20 min at 95-100° C. and thenblocked with 10% normal goat serum (Vector Laboratories, Burlingame,Calif.). The sections were then incubated with rabbit antiserum to humanGIP (Cedarlane Laboratories, Ontario, Canada) at a 1:200 dilution orguinea pig anti-swine insulin (DakoCytomation, Carpinteria, Calif.) at a1:100 dilution for 1 h at room temperature. After washing sections wereincubated for 1 h with either goat anti-rabbit Alexa 488 or goatanti-guinea pig Alexa 555 (Molecular Probes, Eugene, Oreg.) at a 1:600dilution to detect the presence of either insulin or GIP respectively.Finally, the slides were mounted by Vectashield Mounting Medium withDAPI (Vector Laboratories) and viewed using a Leica DMIRB microscope(Leica Microsystems, Richmond Hill, Ontario) equipped with aPhotometrics CoolSNAP camera (Roper Scientific, Tucson, Ariz.) andMetaMorph 7.0 imaging software (PerkinElmer LAS Canada, Woodbridge,Ontario).

Results: The human GIP promoter was used to target human insulinexpression to K-cells in pre-clinical studies using pigs. The human GIPpromoter-linked human insulin cDNA expression cassette with attBsequence and ΦC31 integrase expression plasmid (pCMV-INT) were packagedwith small molecular weight chitosan C(24,98) (NP=40, DNAconcentration=75 ug/ml) and delivered by injection into a demarcatedregion in the pig duodenum. Furthermore, the human GIP promoter-linkedhuman insulin cDNA expression cassette was also packaged in aVSVG-pseudotyped FIV vector and delivered by injection in order tocompare the gene transfer efficiency between the two vectors. Seven daysafter vector administration, biopsies from the demarcated area wereobtained for quantitative RT-PCR with a human insulin specific primerand probe set and immunostaining with antibodies specific for humaninsulin and GIP.

(FIG. 12) Quantitative RT-PCR analysis of biopsy samples from bothchitosan/DNA polyplex treated and VSV-G pseudotyped FIV vector treatedpig duodenum provided detectable human insulin mRNA. This data showsthat the human GIP promotor is active in the pig duodenum. Furthermore,human insulin mRNA levels appeared to be almost 10 fold higher in thechitosan/DNA polyplex treated pig when compared to the FIV treated pigdemonstrating the ability of chitosan/DNA polyplex to act as anefficient gene delivery vector to the pig duodenum.

Distinct cells immunoreactive for human insulin were detected in mucosalcells of the duodenum. These immunopositive cells exhibit a ‘flask-like’shape, which resemble the classical feature of enteroendocrine cells. Inaddition, double immunofluorescence examination revealed co-localizationof insulin with GIP in duodenal cells suggesting that the human GIPpromoter used in our vector correctly targets insulin expression toK-cells in pigs (data not shown).

Luminal Delivery in Pig with Different Pre-Wash Conditions

Chitosan/DNA polyplexes were prepared as described above.

Care of Yorkshire and Yucutan pigs is described above.

Pigs were fasted overnight and pre-medicated by intramuscular injectionof a mixture of atropine (0.04-1.00 mg/kg), ketamine hydrochloride(10-20 mg/kg), and rompun (0.1 mg/kg) by ARU staff. Subsequently,endotracheal intubation was performed on the animal for the delivery of2% isoflurane, which was maintained throughout the entire procedure.Ringer's lactate was administered by intravenous catheterization forvolume replacement. Pigs weighed between 29 and 51 kg at the time ofvector delivery.

A fiber-optic endoscope (Olympus, model CF-1T10L) attached to a videocamera system (Visera OTV-S7V) was used to access the duodenum. Theendoscope contains a working channel through which the biopsy tool,clipping device and catheters were placed. Biopsies were collected indry ice and later transferred to −80° C. Three haemoclips were placedusing the Rotatable Clip Fixing Device (Olympus) to mark the targetregion for virus delivery. Buscopan (0.3 mg/kg IV) was administered tosuppress peristalsis via the IV injection port in the catheter line. Onedose was given at the start of the wash procedure and a second dose wasadministered just prior to delivery of the viral vector solution.

Pre-treatment of duodenum: For all animals the duodenum was treated with40 ml of either 5% N-acetylycysteine (NAC, pH 7.0) for 10 minutes toremove the mucin layer, 19 mM Sodium acetate pH 5 to adjust the pH ofthe surrounding environment, a combination of NAC and Sodium acetate, orsaline alone. All pre-treatment solutions were delivered with the HobbsMistifier spray catheter (REF 2190).

Vector Administration: After the endoscope entered the duodenum, thetreatment site (−5 cm) was marked using duodenal clips (Olympus Canada,HX-600-090L) were placed via the open channel of the endoscope. Thepre-treatment solutions were then sprayed via a radial spray catheterthroughout the entire treatment site. Following pre-treatment, 20 ml ofChitosan/DNA polyplex (DNA concentration=75 ug/ml) containing bothpCMV-SEAP-3×FLAG and pCMV-INT plasmids (5:1 volume ratio) were deliveredto the treatment site with a Mystifier catheter (REF 2190).

Tissue collection: Seven days after delivery, pigs were pre-medicatedand anesthetized as described above. The abdomen was surgically openedas described above to locate the pylorus and proximal region of theduodenum. Clamps were placed across the pyloric sphincter and 25 cmdistal to the pylorus to clamp off the duodenum. Blood vessels adjacentto the duodenum were clamped and then severed. The clamped duodenalsections were incised and the segment removed from the abdomen andplaced into ice-cold saline for washing. Following clamp removal, theduodenum was cut open longitudinally, washed with fresh ice-cold salineand pinned to a cutting board. A scalpel was used to dissect theproximal and distal regions to remove a marked 2.5 mm outer margin toensure all treated tissue is harvested. The resultant tissue segment wassectioned into 2.8 cm sections. Each of these sections was then cut into2 for both RT-Q-PCR and IHC. Tissue samples for RT-qPCR were submergedin TRIzol reagent (Invitrogen Canada Inc; Burlington, ON), thensnap-frozen in dry ice. Tissue samples for IHC were harvested and fixedin the 4% parafolmaldehyde.

Real-time quantitative RT-PCR(RT-q-PCR): Tissue in TRIzol reagent werehomogenized using a ultra-turrax T8 homogenizer (IKA Works Inc.;Wilmington, N.C.). Total tissue RNA was isolated and purified accordingto the manufacture's protocol.

Each RNA sample was first digested by DNase I (Invitrogen) to remove anyDNA contamination. To each 1 ug sample of RNA, 1 μL 10× DNase I ReactionBuffer, 1 μL DNase I and RNase/DNase free water were added to a finalvolume of 10 μL. The tubes were incubated for 15 minutes at roomtemperature and then the DNase I was inactivated by addition of 1 μL of25 mM EDTA. The samples were heated for 10 minutes at 65° C.

Reverse Transcription: First-Strand cDNA was synthesized by usingSuperScript™ II Reverse Transcriptase (Invitrogen). Following DNAdigestion, 1 μL each of Random Primer (Invitrogen) and 10 mM dNTP mix(Invitrogen) were added to each tube, which was heated 65° C. for 5minutes, then put on ice. To each tube, the following were added: 4 μLof 5× first-Strand Buffer (Invitrogen), 2 μL of 0.1M DTT, and 1 μL ofRNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen), and the tubeswere incubated at 42° C. for 2 minutes. 1 μL of SuperScript™ II was thenadded and the tubes were incubated at 42° C. for 50 minutes. Finally,the reaction was terminated by heating at 70° C. for 15 minutes.

Real-Time PCR: Pair of PCR primers and a probe were designed to amplifya portion of the marker gene SEAP and 18S primers were used to normalizethe cDNA quantity of 18S and SEAP between tissues from the experimentaland control groups. The sequences of the SEAP primers were5′-ACATGTGCCAGACAGTGGAGC-3′(Forward) and5′-TCTGGAAGTTGCCCTTGACC-3′(Reverse) (IDT Inc.; Coralville, Iowa). TheSEAP probe was 6FAMCAG CCACGGCCTACCTGTGCG MGBNFQ (Applied Biosystems;Foster City, Calif.). 18S primer and probe, TaqMan Master Mix were alsopurchased from Applied Biosystems. All samples were tested for both SEAPgene and 18S. Working in a lamina flow hood, duplicate samples wereloaded into a MicroAmp® optical 96-well reaction plate (AppliedBiosystems). A 10 μL sample of cDNA (contained 0.3 ug of original RNA),0.25 uL of probe, 12.5 μL TaqMan Master Mix, 1.125 μL of each SEAPprimer or 10 uL of sample cDNA (contained 0.5 ng of original RNA), 1.25uL of 18S primer and probe and 1.25 μL RNase/DNase free water, and 12.5uL of TaqMan Master Mix were added to each well. All samples were run inan ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Thedata were normalized by the 18S and the Log 2 Fold Change expressed therelative quantitative difference between experimental group and thecontrol animals.

Results (data not shown): This study was carried out to identify aneffective method for delivering gene vectors to the pig duodenum. Fourpre-wash conditions were used including 5% N-acetylcysteine (NAC), 19 mMSodium Acetate (NaOAc) buffer pH5.0, both 5% NAC and 10 mM NaOAc andsaline alone prior to chitosan/DNA polyplex administration. The RT-q-PCRresult from pigs exposed to various pre-washing condition demonstratedthat 5% NAC pre-washing as well as combination of 5% NAC and 19 mM NaOAcpH5 buffer produced most efficient gene transfer. Since there is nosignificant statistical difference between the above two pre-washingcondition, we concluded that 5% NAC pre-washing alone is sufficient infacilitating chitosan/DNA polypex to transduce pig duodenum. Pre-washwith saline alone gave a negative result demonstrating that saline washdid not enhance gene transduction in the duodenum.

Pig Matrix Study Demonstrates Low Molecular Weight ChitosanNanoparticles More Robust in Gene Delivery

Chitosan/DNA polyplexes were prepared as described above.

Care of Yorkshire and Yucutan pigs is described above.

Manipulation of pigs is described above under the heading Human GIPpromoter-driven expression of human insulin in pig duoedenum. NACpre-wash, as described above, was used for all of the experiments to aidin gene transduction.

Results (FIG. 13): DNA-chitosan polyplexes C(24,98) at NP20, 40, 60 andC(65,98) at NP5, 20, 30 were delivered to the duodenal cells to testwhich polyplexes provide the greatest gene transduction. The RT-q-PCRresult from pigs exposed to various DNA-chitosan polyplexes showed thatthe 24mer produced the greatest gene transfer, and there was nostatistically significant difference between the various N:P ratiostested in 24mer preparations. The 65mer was also capable of genetransfer to a lesser extent. We can conclude that C(24,98) provides themost efficient gene transfer.

Oral Delivery of Chitosan/DNA Polyplex to Mouse Stomach and Duodenum

This experiment was conducted to investigate whether chitosan-packagedplasmid DNA delivered orally to mice could lead to gene transfer to andgene expression in mucosal cells of the stomach. A plasmid carrying theGIP promoter-linked human insulin gene construct or a E1Fa promoterlinked SEAP gene was mixed with a chitosan (MW: 3.9 kD, Degree ofdeacetylation=98%) at a N:P ratio of 40:1. The polyplex was packaged andcharacterized as described above.

Overnight fasted mice were fed 0.5 mL of a suspension containingchitosan-packaged DNA polyplex (at [DNA]=75 μg/ml) in a single bolus viaa feeding tube. Four hours after vector delivery, treated animals werereturned to their cages with free access to food and water. Two daysafter oral feeding of the polyplex, animals were sacrificed and theirstomach mucosa were collected by tissue scraping. The levels of insulinor SEAP mRNA were quantified by a standard reverse-transcriptionreal-time quantitative PCR assay.

As show in FIG. 14, simple oral administration of chitosan-packaged DNAparticles results in successful transfection and expression of insulinand SEAP gene in stomach mucosa, establishing that transfection of gutmucosal cells in vivo can be achieved by oral administration of anon-viral gene vector.

Physicochemical Characteristics

Particle Sizing

Particle size measurements were made using a Zetasizer Nano lightscattering instrument (Malvern Instruments ZEN 3600). In general,samples were undiluted and loaded into a disposable cuvette (Plastibrand759075D). The Zetasizer was programmed to incubate the sample for 3minutes at 25° C. prior to triplicate 3-minute measurements. Z-averageand polydispersity (PDI) were reported. The Zetasizer was alsoprogrammed to account for the composition of the samples with regards toviscosity and refractive index.

Zeta Potential

Zeta potential measurements were made using a Zetasizer Nano lightscattering instrument (Malvern Instruments ZEN 3600). In general,samples were undiluted or diluted 5 or 10 fold in 10 mM NaCl and loadedinto a Zetasizer folded capillary cell (Malvern Instruments DTS 1060).The Zetasizer was programmed to incubate the sample for 3 minutes at 25°C. prior to measurement. The Zetasizer was programmed to account for thefinal composition of the samples with regards to viscosity anddielectric constant.

1. A composition comprising a chitosan-based nanoparticle, saidchitosan-based nanoparticle comprising (i) a plurality of chitosanpolymers having an average molecular weight between 3 kDa and 50 kDa,and (ii) encapsulating a therapeutic construct, wherein said therapeuticconstruct comprises a therapeutic nucleic acid encoding a therapeuticprotein operably linked to an expression control region that isfunctional in a gut mucosal cell, wherein said chitosan-basednanoparticle is stable in an agarose gel retention assay and is capableof producing an increase in the systemic level of said encodedtherapeutic protein when delivered to the gut mucosa. 2-4. (canceled) 5.The composition according to claim 1, wherein said plurality of chitosanpolymers has an average molecular weight less than 25 kDa.
 6. Thecomposition according to claim 1, wherein said chitosan-basednanoparticle has an N:P ratio between 10:1 and 90:1.
 7. The compositionaccording to claim 1, wherein said chitosan-based nanoparticle has adiameter less than 225 nm. 8-11. (canceled)
 12. The compositionaccording to claim 1, wherein said expression control region comprises agut-specific promoter.
 13. (canceled)
 14. (canceled)
 15. (canceled) 16.(canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. Thecomposition according to claim 1, wherein said plurality of chitosanpolymers has an average molecular weight between 3 kDa and 10 kDa. 21.The composition according to claim 1, wherein said plurality of chitosanpolymers has an average molecular weight between 3 kDa and 6 kDa.